http://2010.igem.org/wiki/index.php?title=Special:Contributions/Litagaki&feed=atom&limit=50&target=Litagaki&year=&month=2010.igem.org - User contributions [en]2024-03-28T17:03:11ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T03:50:22Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct (minus background) <br/><br />
gaggaggtttattactagatgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcgctgggcgcttttccgcccgggg<br />
cgctggcgcgcaacccgattgccggcattaacggtaaaaccacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcaggt<br />
agtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacgcgcatatgcgccaggggcgggagcggcgtcagcctggtcaat<br />
gacccgcaacggattgtaaaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaacaggcttaccaggaaatcgcgg<br />
cgaaaatgaatgccatcaaagcgcagcatggccccgagagcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacggc<br />
ctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccattgcggcaaaagtgatgatgggcggcgatctggcgatggatatc<br />
gctaacacgcgctatctggtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatgaccgcgcaggagaagggggcca<br />
aaatggtgagcttcgatccgcgtttgtcgatattttccagcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcgat<br />
gtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttataccagcggatttgaacagttagcacaggcggtaaaagagacgacg<br />
ccggaatgggccgccgcgcaggccgatgtcccagccgacgttattgtccgggtgacacgcgaactggctgcctgcgcgcctcacgctattgtcagtcctg<br />
gtcatcgcgcgacgttctcgcaggaagagatcgatatgcggcgtatgatttttacgcttaatgtgctgctcggtaatattgagcgcgaaggcgggctata<br />
tcagaaaaaaaacgcgtctgtttacaataaactggccggagaaaaggtcgcgccaacgctggcgaaactcaacattaaaaatatgccgaaaccgacggcg<br />
caacgcatcgatttggtcgcaccgcagtttaaatatatcgccgctggcggcggcgtggtgcaaagcattattgactcggcgttaacccagaagccttacc<br />
cgataaaggcgtggattatgtcgcggcataatcctttccagaccgtcacctgtcgttcggacctggtaaaaaccgttgagcaactggatctggtggtcag<br />
ctgcgatgtctatttgagcgagagcgcggcatatgccgactatctgctgccggaatgcacctatctcgaacgggacgaagaggtatccgatatgtcggga<br />
ctgcacccggcttacgctctgcgccaacaggtcgtagagccgattggcgaggcgcgtccgagttggcaaatctggaaagaacttggcgagcagttgggat<br />
tagggcagtactatccgtggcaggatatgcagacgcgccaactctatcagttgaacggcgaccatgccttagcgaaggaactgcgacagaaagggtatct<br />
cgaatggggcgttccgctgctattacgcgaaccagaatccgttcgtcagtttacggcgcgttaccccggcgctatcgcgacggacagtgacaacacctat<br />
ggcgaacagcttcgcttcaaatcgccctccggcaaaatcgaactttattccgcaaccctggaggaattgctccctggctacggcgttccgcgcgttcgtg<br />
actttgcgctgaaaaaagagaatgagctttacttcattcagggcaaggtggccgtgcataccaatggcgcgacgcagtacgtacctttactcagcgagct<br />
aatgtgggataacgcggtctgggttcatccgcaaacggcgcaagaaaaaggcattaagaccggcgatgagatctggctggaaaatgccacgggtaaagag<br />
aaaggtaaggcgctggtgacgcccggtatccgcccggacacgctttttgtctatatgggatttggcgctaaagctggggccaaaacggcggcgacgacac<br />
acggtatccactgcggaaatttactgccgcacgtgacgagtccggtatccggtacggtagtgcataccgcaggcgtgacgctgagccgggcatgaatgaa<br />
tcatttaacgaatcagtacgtcatgctgcatgatgaaaaacgttgtatcggctgccaggcgtgtaccgttgcctgcaaagtgcttaatgacgtaccggag<br />
ggatttagccgcgtacaggtacaaattcgcgcccctgaacaggcatccaacgcactaacccattttcaatttgtccgcgtctcctgtcagcactgtgaaa<br />
atgcgccatgtgtcagcgtttgtcctaccggagcgtcatatcgtgatgaaaacgggatcgtgcaggtggataaatcgcgctgtattggctgcgattattg<br />
tgttgccgcgtgtcctttccatgtgcgctatttgaatccgcaaaccggtgtcgccgacaagtgtaacttctgcgccgacacgcggttggcggctggccag<br />
tctccggcgtgcgtatccgtttgcccaacggacgcgctgaaattcggcagactggatgagagcgagatccagcgctgggtcggtcagaaagaggtctatc<br />
gccagcaggaggcgcgtagcggcgcggtcagtctgtaccgtcgtaaagaagtccatcaggagggtaaagcatgaatgaatactatctggggagcggaact<br />
acattatgcgccagattattggccgctgtggttaatttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcgccgg<br />
atgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctggcgattcgccgctggcattggggaaatgcgttactgtttgttt<br />
tattactgttaagcggtttatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtggttttgccttactggctttctg<br />
ggtcgggtttgtgctgatcaacctcaccacaggtaacgggcgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgtttt<br />
tacctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagttcaatccactgcaacaactggcatatctggcgattatgt<br />
acgcgctggtaccgctgttaatcatcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgctgcatatggcgcttgc<br />
tatcatcggcttactgtttatttgcgcgcatctctatctgtgtactcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgt<br />
catcgtaccgcgccgcgcggggataagtccgccgtctgatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt<br />
atctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata <br/><br/><br />
<br />
>Forward sequence of ligation sample <br/><br />
nnnnnngnnttnnnnnnnnnnntaggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcgcggcc<br />
gcttctagagaggaggtttatatgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcgctgggcgcttttccgcccg<br />
gggcgctggcgcgcaacccgattgccggcattaacggtaaaaccacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgca<br />
ggtagtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacgcgcatatgcgccaggggcgggagcggcgtcagcctggtc<br />
aatgacccgcaacggattgtaaaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaacaggcttaccaggaaatcg<br />
cggcgaaaatgaatgccatcaaagcgcagcatggccccgagagcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctac<br />
ggcctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccattgcggcaaaagtgatgatgggcggcgatctggcgatggat<br />
atcgctaacacgcgctatctggtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatgaccgcgcaggagaaggggg<br />
ccaaaatggtgagcttcgatccgcgtttgtcgatattttccagcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggc<br />
gatgtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttataccagcggatttgaacagttagcacaggcggtaaaagagacg<br />
acgccggaatgggccnngcgcaggccgatgtn<br />
<br/> <br/><br />
>Reverse sequence of ligation sample <br/><br />
actatctggggangcggaactacattatgcgccagattattggccgctgnggttaatttacgcaggcgtcgtggtgctgctcatgcttgttgggctggtt<br />
atccatgcgttattgcgccggatgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctggcgattcgccgctggcattggg<br />
gaaatgcgttactgtttgttttattactgttaagcggtttatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtgg<br />
ttttgccttactggctttctgggtcgggtttgtgctgatcaacctcaccacaggtaacgggcgtcactatcgggtaaatttttccggactggtaacgcgc<br />
tgcatacgccagacgcgtttttacctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagttcaatccactgcaacaac<br />
tggcatatctggcgattatgtacgcgctggtaccgctgttaatcatcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgct<br />
ggtgctgcatatggcgcttgctatcatcggcttactgtttatttgcgcgcatctctatctgtgtactcttggcgacacgccgggacaaattttccgtagc<br />
atggttgacggctatcatcgtcatcgtaccgcgccgcgcggggataagtccgccgtctgatactagagccaggcatcaaataaaacgaaaggctcagtcg<br />
aaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatac<br />
tagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcc<br />
tcgctcactgactcgctgcgctcggtcgtngnnnnnnnnnn <br />
<!------------- atgc -------------><br />
</div><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T03:48:57Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct (minus background) <br/><br />
gaggaggtttattactagatgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcgctgggcgcttttccgcccgggg<br />
cgctggcgcgcaacccgattgccggcattaacggtaaaaccacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcaggt<br />
agtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacgcgcatatgcgccaggggcgggagcggcgtcagcctggtcaat<br />
gacccgcaacggattgtaaaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaacaggcttaccaggaaatcgcgg<br />
cgaaaatgaatgccatcaaagcgcagcatggccccgagagcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacggc<br />
ctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccattgcggcaaaagtgatgatgggcggcgatctggcgatggatatc<br />
gctaacacgcgctatctggtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatgaccgcgcaggagaagggggcca<br />
aaatggtgagcttcgatccgcgtttgtcgatattttccagcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcgat<br />
gtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttataccagcggatttgaacagttagcacaggcggtaaaagagacgacg<br />
ccggaatgggccgccgcgcaggccgatgtcccagccgacgttattgtccgggtgacacgcgaactggctgcctgcgcgcctcacgctattgtcagtcctg<br />
gtcatcgcgcgacgttctcgcaggaagagatcgatatgcggcgtatgatttttacgcttaatgtgctgctcggtaatattgagcgcgaaggcgggctata<br />
tcagaaaaaaaacgcgtctgtttacaataaactggccggagaaaaggtcgcgccaacgctggcgaaactcaacattaaaaatatgccgaaaccgacggcg<br />
caacgcatcgatttggtcgcaccgcagtttaaatatatcgccgctggcggcggcgtggtgcaaagcattattgactcggcgttaacccagaagccttacc<br />
cgataaaggcgtggattatgtcgcggcataatcctttccagaccgtcacctgtcgttcggacctggtaaaaaccgttgagcaactggatctggtggtcag<br />
ctgcgatgtctatttgagcgagagcgcggcatatgccgactatctgctgccggaatgcacctatctcgaacgggacgaagaggtatccgatatgtcggga<br />
ctgcacccggcttacgctctgcgccaacaggtcgtagagccgattggcgaggcgcgtccgagttggcaaatctggaaagaacttggcgagcagttgggat<br />
tagggcagtactatccgtggcaggatatgcagacgcgccaactctatcagttgaacggcgaccatgccttagcgaaggaactgcgacagaaagggtatct<br />
cgaatggggcgttccgctgctattacgcgaaccagaatccgttcgtcagtttacggcgcgttaccccggcgctatcgcgacggacagtgacaacacctat<br />
ggcgaacagcttcgcttcaaatcgccctccggcaaaatcgaactttattccgcaaccctggaggaattgctccctggctacggcgttccgcgcgttcgtg<br />
actttgcgctgaaaaaagagaatgagctttacttcattcagggcaaggtggccgtgcataccaatggcgcgacgcagtacgtacctttactcagcgagct<br />
aatgtgggataacgcggtctgggttcatccgcaaacggcgcaagaaaaaggcattaagaccggcgatgagatctggctggaaaatgccacgggtaaagag<br />
aaaggtaaggcgctggtgacgcccggtatccgcccggacacgctttttgtctatatgggatttggcgctaaagctggggccaaaacggcggcgacgacac<br />
acggtatccactgcggaaatttactgccgcacgtgacgagtccggtatccggtacggtagtgcataccgcaggcgtgacgctgagccgggcatgaatgaa<br />
tcatttaacgaatcagtacgtcatgctgcatgatgaaaaacgttgtatcggctgccaggcgtgtaccgttgcctgcaaagtgcttaatgacgtaccggag<br />
ggatttagccgcgtacaggtacaaattcgcgcccctgaacaggcatccaacgcactaacccattttcaatttgtccgcgtctcctgtcagcactgtgaaa<br />
atgcgccatgtgtcagcgtttgtcctaccggagcgtcatatcgtgatgaaaacgggatcgtgcaggtggataaatcgcgctgtattggctgcgattattg<br />
tgttgccgcgtgtcctttccatgtgcgctatttgaatccgcaaaccggtgtcgccgacaagtgtaacttctgcgccgacacgcggttggcggctggccag<br />
tctccggcgtgcgtatccgtttgcccaacggacgcgctgaaattcggcagactggatgagagcgagatccagcgctgggtcggtcagaaagaggtctatc<br />
gccagcaggaggcgcgtagcggcgcggtcagtctgtaccgtcgtaaagaagtccatcaggagggtaaagcatgaatgaatactatctggggagcggaact<br />
acattatgcgccagattattggccgctgtggttaatttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcgccgg<br />
atgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctggcgattcgccgctggcattggggaaatgcgttactgtttgttt<br />
tattactgttaagcggtttatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtggttttgccttactggctttctg<br />
ggtcgggtttgtgctgatcaacctcaccacaggtaacgggcgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgtttt<br />
tacctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagttcaatccactgcaacaactggcatatctggcgattatgt<br />
acgcgctggtaccgctgttaatcatcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgctgcatatggcgcttgc<br />
tatcatcggcttactgtttatttgcgcgcatctctatctgtgtactcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgt<br />
catcgtaccgcgccgcgcggggataagtccgccgtctgatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt<br />
atctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata <br/><br/><br />
<br />
>Forward sequence of ligation sample <br/><br />
nnnnnngnnttnnnnnnnnnnntaggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcgcggccgcttctagagaggaggtttatatgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcgctgggcgcttttccgcccggggcgctggcgcgcaacccgattgccggcattaacggtaaaaccacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcaggtagtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacgcgcatatgcgccaggggcgggagcggcgtcagcctggtcaatgacccgcaacggattgtaaaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaacaggcttaccaggaaatcgcggcgaaaatgaatgccatcaaagcgcagcatggccccgagagcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacggcctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccattgcggcaaaagtgatgatgggcggcgatctggcgatggatatcgctaacacgcgctatctggtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatgaccgcgcaggagaagggggccaaaatggtgagcttcgatccgcgtttgtcgatattttccagcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcgatgtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttataccagcggatttgaacagttagcacaggcggtaaaagagacgacgccggaatgggccnngcgcaggccgatgtn<br />
<br/> <br/><br />
>Reverse sequence of ligation sample <br/><br />
actatctggggangcggaactacattatgcgccagattattggccgctgnggttaatttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcgccggatgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctggcgattcgccgctggcattggggaaatgcgttactgtttgttttattactgttaagcggtttatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtggttttgccttactggctttctgggtcgggtttgtgctgatcaacctcaccacaggtaacgggcgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgtttttacctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagttcaatccactgcaacaactggcatatctggcgattatgtacgcgctggtaccgctgttaatcatcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgctgcatatggcgcttgctatcatcggcttactgtttatttgcgcgcatctctatctgtgtactcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgtcatcgtaccgcgccgcgcggggataagtccgccgtctgatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctcggtcgtngnnnnnnnnnn <br />
<!------------- atgc -------------><br />
</div><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T03:32:30Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct (minus background) <br/><br />
gaggaggtttattactagatgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcgctgggcgcttttccgcccgggg<br />
cgctggcgcgcaacccgattgccggcattaacggtaaaaccacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcaggt<br />
agtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacgcgcatatgcgccaggggcgggagcggcgtcagcctggtcaat<br />
gacccgcaacggattgtaaaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaacaggcttaccaggaaatcgcgg<br />
cgaaaatgaatgccatcaaagcgcagcatggccccgagagcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacggc<br />
ctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccattgcggcaaaagtgatgatgggcggcgatctggcgatggatatc<br />
gctaacacgcgctatctggtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatgaccgcgcaggagaagggggcca<br />
aaatggtgagcttcgatccgcgtttgtcgatattttccagcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcgat<br />
gtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttataccagcggatttgaacagttagcacaggcggtaaaagagacgacg<br />
ccggaatgggccgccgcgcaggccgatgtcccagccgacgttattgtccgggtgacacgcgaactggctgcctgcgcgcctcacgctattgtcagtcctg<br />
gtcatcgcgcgacgttctcgcaggaagagatcgatatgcggcgtatgatttttacgcttaatgtgctgctcggtaatattgagcgcgaaggcgggctata<br />
tcagaaaaaaaacgcgtctgtttacaataaactggccggagaaaaggtcgcgccaacgctggcgaaactcaacattaaaaatatgccgaaaccgacggcg<br />
caacgcatcgatttggtcgcaccgcagtttaaatatatcgccgctggcggcggcgtggtgcaaagcattattgactcggcgttaacccagaagccttacc<br />
cgataaaggcgtggattatgtcgcggcataatcctttccagaccgtcacctgtcgttcggacctggtaaaaaccgttgagcaactggatctggtggtcag<br />
ctgcgatgtctatttgagcgagagcgcggcatatgccgactatctgctgccggaatgcacctatctcgaacgggacgaagaggtatccgatatgtcggga<br />
ctgcacccggcttacgctctgcgccaacaggtcgtagagccgattggcgaggcgcgtccgagttggcaaatctggaaagaacttggcgagcagttgggat<br />
tagggcagtactatccgtggcaggatatgcagacgcgccaactctatcagttgaacggcgaccatgccttagcgaaggaactgcgacagaaagggtatct<br />
cgaatggggcgttccgctgctattacgcgaaccagaatccgttcgtcagtttacggcgcgttaccccggcgctatcgcgacggacagtgacaacacctat<br />
ggcgaacagcttcgcttcaaatcgccctccggcaaaatcgaactttattccgcaaccctggaggaattgctccctggctacggcgttccgcgcgttcgtg<br />
actttgcgctgaaaaaagagaatgagctttacttcattcagggcaaggtggccgtgcataccaatggcgcgacgcagtacgtacctttactcagcgagct<br />
aatgtgggataacgcggtctgggttcatccgcaaacggcgcaagaaaaaggcattaagaccggcgatgagatctggctggaaaatgccacgggtaaagag<br />
aaaggtaaggcgctggtgacgcccggtatccgcccggacacgctttttgtctatatgggatttggcgctaaagctggggccaaaacggcggcgacgacac<br />
acggtatccactgcggaaatttactgccgcacgtgacgagtccggtatccggtacggtagtgcataccgcaggcgtgacgctgagccgggcatgaatgaa<br />
tcatttaacgaatcagtacgtcatgctgcatgatgaaaaacgttgtatcggctgccaggcgtgtaccgttgcctgcaaagtgcttaatgacgtaccggag<br />
ggatttagccgcgtacaggtacaaattcgcgcccctgaacaggcatccaacgcactaacccattttcaatttgtccgcgtctcctgtcagcactgtgaaa<br />
atgcgccatgtgtcagcgtttgtcctaccggagcgtcatatcgtgatgaaaacgggatcgtgcaggtggataaatcgcgctgtattggctgcgattattg<br />
tgttgccgcgtgtcctttccatgtgcgctatttgaatccgcaaaccggtgtcgccgacaagtgtaacttctgcgccgacacgcggttggcggctggccag<br />
tctccggcgtgcgtatccgtttgcccaacggacgcgctgaaattcggcagactggatgagagcgagatccagcgctgggtcggtcagaaagaggtctatc<br />
gccagcaggaggcgcgtagcggcgcggtcagtctgtaccgtcgtaaagaagtccatcaggagggtaaagcatgaatgaatactatctggggagcggaact<br />
acattatgcgccagattattggccgctgtggttaatttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcgccgg<br />
atgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctggcgattcgccgctggcattggggaaatgcgttactgtttgttt<br />
tattactgttaagcggtttatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtggttttgccttactggctttctg<br />
ggtcgggtttgtgctgatcaacctcaccacaggtaacgggcgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgtttt<br />
tacctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagttcaatccactgcaacaactggcatatctggcgattatgt<br />
acgcgctggtaccgctgttaatcatcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgctgcatatggcgcttgc<br />
tatcatcggcttactgtttatttgcgcgcatctctatctgtgtactcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgt<br />
catcgtaccgcgccgcgcggggataagtccgccgtctgatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt<br />
atctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata <br/><br/><br />
<br />
>Forward sequence of ligation sample <br/><br />
<br />
>Reverse sequence of ligation sample <br/><br />
<!------------- atgc -------------><br />
</div><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T03:29:34Z<p>Litagaki: Undo revision 207250 by Litagaki (Talk)</p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T03:23:41Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_9Team:Yale/Our Project/Notebook/Week 92010-10-28T03:12:36Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body onLoad="resettoggle()"><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 9 <br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
Monday 8/2--Analysis of second ligation transformants<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday-------------><br />
<h4> Work with transformants from attempt #1 to complete second ligation </h4><br />
<ul><br />
<li>Chose seven colonies from the 2:1 insert:vector ligation plate (colonies #1-#7) and seven from the 5:1 ligation plate (colonies #8-#14) to analyze. Used each to inoculate a liquid culture, streak an index plate, and run a colony PCR reaction (after appropriate lysing in water).</li><br />
<li>Each PCR reaction mixture contained 2 uL of VF2 primer, 2 of VR primer, 9.8 uL of water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL of dNTPs, and 0.2 uL of polymerase. They were run on the 'VR' thermocycler protocol</li><br />
<li> The PCR products were then run on a 1.0% agarose gel at 90 V vs. a 1 kb ladder.</li><br />
Loaded ladder at the far left and samples 1 through 14 from left to right in order. Only sample 6 is even close the size range of the desired ligation product (8.3 kb) and even it is still at least a kb too small to be the right construct. <br/><br />
<i> This day's labwork is also recorded on pages 85 and 86 of the hard copy lab notebook. </i><br />
<!------------- Monday-------------><br />
</div><br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_9Team:Yale/Our Project/Notebook/Week 92010-10-28T03:00:09Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body onLoad="resettoggle()"><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 9 <br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
Monday 8/2--Analysis of second ligation transformants<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday-------------><br />
<i> This day's labwork is also recorded on pages 85 and 86 of the hard copy lab notebook. </i><br />
<!------------- Monday-------------><br />
</div><br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_9Team:Yale/Our Project/Notebook/Week 92010-10-28T02:59:15Z<p>Litagaki: New page: __NOTOC__ {{:Team:Yale/Templates/Yale_Header_Project}} <html> <body onLoad="resettoggle()"> <div id="main-content"> <div id="left-col"> <h5> our project </h5> <p> <ul id="proj-nav"> <li><a...</p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body onLoad="resettoggle()"><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 9 <br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
Monday 8/2--Analysis of second ligation transformants<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday-------------><br />
<i> This day's labwork is also recorded on pages 85 and 86 of the hard copy lab notebook. </i><br />
<!------------- Monday-------------><br />
</div><br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-28T02:53:50Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract"> gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" /><br />
</div><br />
<br /><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a9/Yale-self-ligation.jpg" /><br />
</div><br />
<br /><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
</ul><br />
<h4>Ligation using serial digestion products </h4><br />
<ul><br />
<li>Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the <a href=" https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> standard protocol </a>, then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015. </li><br />
<li> Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA </li><br />
<b>T4 ligase control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water <br/><br />
<b>T4 ligase 2:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water <br/><br />
<b>T4 ligase 6:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC <br/><br />
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/><br />
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/><br />
<br />
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li><br />
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/><br />
<b>2:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water <br/><br />
<b>2:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water <br/><br />
<b>6:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC<br/><br />
<b>6:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC <br/><br />
Molar ratios are approximate.<br/><br />
<li> All ligations were allowed to run overnight at 16˚C. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--Ligation Transformations<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<b> Q02141 transformation </b> <br/><br />
<ul><br />
<li><br />
No growth was observed on the Q04121 plates--may need to find better competent cells. </li><br />
</ul><br />
<h4> Transformation of Varied Ligation Efforts </h4><br />
<ul><br />
<li> Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C. </li><br />
<li> After previous ligation troubles, have invested in commercial grade ultracompetent cells. Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a>, also transforming the pUC19 standard that came with the cells. All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--Results of ligation transformations<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Ligation Transformants </h4><br />
<ul><br />
<li> The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable. The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all. In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate. </li><br />
<li> Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and mixing a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the resulting PCR reaction on the "VR" protocol of the thermocycler. </li><br />
<li> Also started liquid cultures of each of the colonies in ampicillin LB. </li><br />
<li> The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24 </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4> Gel of Colony PCR </h4><br />
<ul><br />
<li> Ran the twenty-four colony PCR reactions from 7/23 on a single massive 1.0% agarose gel vs a 1 kb ladder at 90 V.</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f6/Yale-colony-pcr.jpg" /><br />
</div><br />
<div id="caption"><br />
Ladder is very faint in the leftmost lane and the twenty-four reactions are in order from left to right. Despite the faintness, one can see that samples 5, 12, 14, 15, 20, 21 and 24 appear to have the desired 3.6 kb fragment indication a successful ligation.</div><br />
</ul><br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--Inoculate cultures of likely ligations successes for miniprep on 7/26<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
<b> Inoculation of liquid cultures </b> <br/><br />
<ul><br />
<li> Inoculated liquid ampicillin in LB cultures from the colonies shown to contain successful ligations by the 9/25 gel of colony PCR. Also inoculated a culture of Q04121 for overnight growth, adding kanamycin to a concentration of 50 ng/uL. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
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<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-28T02:52:20Z<p>Litagaki: </p>
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our project<br />
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<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
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<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-le392-ps574.jpg" /><br />
</div><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. Restarted power source, let run, and then visualized the gels </li><br />
<li>Digestion of miniprep shows that all ligation efforts failed--had they succeeded there would have been fragments at 7.8 kb, but as the gel below shows, all the samples run at slightly over 3 kb (ladder rungs are 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 8 kb, & 10 kb).</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/c/cf/Yale-ligation-digest.jpg" /><br />
</div><br />
<li> Efforts to visualize the colony PCR gel failed entirely--maybe forgot to add ethidium bromide? But given above results, it's a moot point. </li><br />
</ul><br />
<h4> Copper Removal Assay Prep </h4><br />
<ul><br />
<li> Want to run an assay measuring whether pSB74 transformants remove copper(II) from their surroundings. If had a large bacterial culture with an intermediate CuSO<sub>4</sub> concentration (say 500 uM), could periodically remove small aliquots, centrifuge out the cells, and spectrophotometrically determine the copper content. </li><br />
<li> Need to determine what wavelength should be used to monitor copper concentration and create a calibration curve relating absorbance at that wavelength to copper concentration. </li><br />
<li> Start by making serial dilutions of CuSO<sub>4</sub> in LB. Made samples of 0.1 M, 10 mM, 1 mM, 100 uM, 10 uM, 1 uM and 0.1 uM concentrations. </li><br />
<li> While spectrum had no clear peak, the CuSO<sub>4</sub> did absorb strongly at 700 nm compared to just LB, so chose that as wavelength to monitor. Attempted to establish curve, but got the odd result that the 10 uM absorbed orders of magnitude more than the 100 uM. Will redo dilutions and try again another day. </li><br />
<li> Inoculated 5 mL liquid cultures of LE392, both with and without pSB74, adding 5 uL of 1000x ampicillin to the transformant culture. Let grow overnight on shaker at 37˚C. </li><br />
<br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
<h4> Digest/Ligation Attempt #5 </h4><br />
<ul><br />
<li> Concerned that ligation components may not be thoroughly digested, so ran an all day digestion of phsABC, which containted 5 uL EcoRI buffer, 0.5 uL 100x BSA, 36.1 uL phsABC (at 27.7 ng/uL for 1 ug total), 1.8 uL EcoRI, 1.8 uL SpeI, and 4.8 uL water. </li><br />
<li> Also concerned about SpeI activity, so ran the following diagnostic SpeI digestion of B0015 that will then be run on a gel versus the circularized plasmid: 5 uL NEB buffer 4, 0.5 uL 100x BSA, 4 uL B0015(1 ug DNA), 3.6 uL SpeI, and 36.9 uL water, let to run for 2 hours at 37˚C before heat killing at 80˚C for twenty minutes </li><br />
<li>Simultaneously digested more B0015 with XbaI according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5">7/8 </a> to ensure there will be enough vector. </li><br />
<li> Purified the XbaI-digested B0015 with a standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>, eluting in 40 uL, and then digested all of the result with 3.6 uL EcoRI, 5 uL EcoRI buffer, 0.5 uL 100x BSA, and 0.9 uL of water, letting it run for two hours. An hour into the digestion, added 1 uL of CIP to the reaction, and after the digestion heat-killed the enzymes with 20 minutes at 80˚C. </li><br />
</ul><br />
<h4>Copper(II) Absorbance Calibration Curve Redo</h4><br />
<ul><br />
<li> Once again carefully make serial dilutions of copper sulfate in LB, creating a series of solutions running from 0.1 M to 1 uM and separated by a power of ten. Absorbances prove to be highly nonlinear again, so research and find that copper(II) concentration cannot directly be measured directly spectrophotometrically. </li><br />
</ul><br />
<h4> Bacterial Survival in Copper Solution </h4><br />
<ul><br />
<li> Retrieve from incubator plates spotted with copper solution cultures from 6/13 copper growth assay. Find that all cultures up to and including 4 mM copper levels survived, both in the transformed and untransformed LE392. Also see a colony were the pSB74 transformant in 50 mM copper was spotted, but as there is no growth at 10 mM, wonder if there was an accidental drip during spotting. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--Confirmation of SpeI activity and EtOH precipitation of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
<ul><br />
<b>Analysis of SpeI activity</b> <br/><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/7/78/Yale-spe1test.jpg" /><br />
</div><br />
<li> Ran the SpeI digest of B0015 on a gel versus the circular B0015 and a 1 kb ladder (leftmost lane). The digested plasmid (rightmost lane) ran slower than the undigested one (middle lane), confirming that SpeI is active and successfully linearized the plasmid. </li><br />
</ul><br />
<b>Ethanol precipitation of ligation components </b> <br/><br />
<ul><br />
<li> Guessing that higher concentrations of insert and vector might improve ligation results, ethanol precipitate the digested samples from 7/14 according to the following <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> protocol </a> </li><br />
<li> In case need more material, started five overnight liquid cultures of each pSB74 and B0015 in Amp LB for morning miniprep. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on page 69 of the hard copy lab notebook.</i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--Experimentation with different treatments of vector & insert DNA prior to ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Stockpiling starting materials </h4><br />
<ul><br />
<li> Miniprepped the overnight cultures of pSB74 and B0015 <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> by vacuum manifold </a> and found the resulting concentrations by nanodrop. The five B0015 samples had concentrations of 83.7, 83.4, 111.7, 130.0, and 103.3 ng/uL while the pSB74 samples had concentration values of 118.7, 129.5, 118.2, 137.3, and 124.9 ng/uL. </li><br />
<li> Set up 8 PCR reactions with pSB74 samples using the "phs50" thermocycler protocol and the DMSO variant of PCR reaction contents from <a href=" https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4"> 6/30 </a>.</li><br />
<li> Also ran four digestions of B0015 with XbaI, each of which included 5 uL NEB buffer 4, 0.5 uL 100x BSA, 3.6 uL XbaI, 7.7 uL B0015 (1 ug worth), and 33.2 uL of water. Let these run for two hours at 37˚C </li><br />
<li> Meanwhile attempted to resuspend ethanol precipitated DNA, but nanodrop showed no DNA present--pellets must have fallen out of tubes when inverted to dry. </li><br />
<li> After XbaI digestion of B0015 skipped PCR purification step (concerned that it was leading to product loss)and simply added 0.5 uL of 5 M NaCl to each digestion so that the buffer solution would have the salt content required by EcoRI. Then added 2 uL of EcoRI to each digestion and let incubate for 2 hours at 37˚C. One hour into this digestion, added 1 uL of CIP to half (two) of the digestion reactions (want to see if it makes a difference). </li><br />
<li> Following the PCR amplification of phsABC, ran the vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR cleanup protocol</a> on four out of the eight reaction solutions and measured the DNA concentrations of the resulting solutions as 99.5, 131.2, 75.5, 36.2, and 26.2 ng/uL. </li><br />
<li> Concerned that phsABC may not be getting digested properly, especially given that its cut sites are so near its ends. While running the required EcoRI/SpeI double digestion, will run in parallel single digestions of phsABC with each EcoRI and SpeI. While the amount cut off makes these digestions impossible to detect directly, if the digestions occur properly the resulting fragments should be able to self-ligate, so it is possible to test for that. </li><br />
<li> The two double digestions had contents as follows: 5 uL EcoRI buffer, 0.5 uL 100x BSA, 10.5 uL phsABC (1 ug DNA), 1.8 uL EcoRI, 1.8 uL SpeI, and 30.4 uL water. </li><br />
<li> The diagnostic single digestions were run with 13.2 uL phsABC (at 75.5 ng/uL, 1 ug), 5 uL EcoRI buffer, 0.5 uL BSA, 27.7 uL water, and 3.6 uL of the relevant enzyme whether EcoRI or SpeI. </li><br />
<li> All of the above digestions were run for eight hours at 37˚C before being heat-killed with 20 minutes at 80˚C. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 70 & 71 of the hard copy lab notebook.</i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
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</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5Team:Yale/Our Project/Notebook/Week 52010-10-28T02:51:19Z<p>Litagaki: </p>
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<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 5 (7/5 -7/11)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/5--Colony PCR of Ligation Attempt #2 Transformants<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4> Colony PCR</h4><br />
<ul><br />
<li>Ran colony PCR of colonies 1B-9B from ligation attempt #2 according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4"> 7/1</a>.</li><br />
<li>Then ran a 1.0% agarose gel of the nine PCR reaction solutions against a 1 kb ladder, but the resulting gel failed to show any evidence of either insert or vector, so will have to rely on sequencing and digests of miniprepped plasmids.</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/Yale-ligation-products.jpg" /><br />
</div><br />
<div id="caption">Gel of colony PCR from ligation attempt #2 Lane 1: 1 kb ladder, Lanes 3-11:PCR products from colonies 1B-9B, in order</div><br />
<br />
<li>Miniprepped liquid cultures grown from colonies of first ligation attempt (colonies 1A-4A) and second ligation attempt (colonies 1B-9B) according to <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard miniprep protocol</a> </li><br />
</ul><br />
<i>This day's activities are also recorded on page 47 and 48 of the hard copy lab notebook. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/6--Analysis of Results of Ligation Attempts 1 & 2<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> More Diagnostics of Ligation Results</h4><br />
<ul><br />
<li>In order to determine success of ligation attempts 1 and 2, digested the miniprepped plasmids 1A-4A and 1B-9B from 7/5 with XbaI and SpeI and then looked for evidence of two fragments, one at 3.2 kb and the other at 3.6 kb. Reactions were conducted for 2 hours at 37°C, with the following contents for each of the thirteen reactions </li><br />
<table><br />
<tr><td><br />
Component </td> <td> Volume<br />
</td> </tr><br />
<tr> <td><br />
Distilled Water </td> <td>6.4 μL<br />
</td> </tr><br />
<tr> <td><br />
10x NEB Buffer 4 </td> <td>2 μL<br />
</td> </tr><br />
<tr> <td><br />
DNA solution </td> <td>10 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB XbaI </td> <td>0.8 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB SpeI </td> <td>0.8 μL<br />
</td> </tr><br />
<tr> <td><br />
Total </td> <td>20 μL<br />
</td></tr><br />
</table><br />
<br />
<li>The diagnostic 1.0% agarose gel was run at 90 V vs. a 1 kb ladder. Each of the thirteen samples gave one diffuse band around 3 kb and no evidence of the 3.6 kb phsABC fragment, so both ligation attempts #1 and #2 were unsuccessful.</li><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/9/93/Yale-digest-gel.jpg" /><br />
</div><br />
<div id="caption">Gel of XbaI and SpeI double digest of plasmids from ligation attempts #1 and #2 Lane 1: 1 kb ladder, Lanes 2-5:1A-4A, Lanes 6-14: 1B-9B </div><br />
<br />
<li>During digestion prepped plasmid samples for sequencing. Each sample contained 3 μL of plasmid soln (average concentration of 96 ng/μL), 2 μL of 4 mM VF2 (forward primer), and 13 μL of distilled water. Intended to follow up with reverse sequencing of more promising samples, but gel results made it a moot point. </li><br />
</ul><br />
<br />
<i>Wetlab work for this day is also recorded on pages 47, 49, and 50 in the hard copy lab notebook. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/7--Triple ligation attempt--promoter + thiosulfate reductase + terminator<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<h4>Ligation Strategy </h4><br />
<ul><br />
<li>Failure of previous two ligation efforts prompted rethinking of ligation strategy. Will attempt a one-step triple ligation that will link phsABC to B0015 and insert them both into a standard single-resistance iGEM background. phsABC will be digested with EcoRI and SpeI, B0015 with XbaI and PstI, and pSB1C3 with EcoRI and PstI, allowing for them all to be ligated together. </li><br />
</ul><br />
<br />
<b>Prep work </b> <br/><br />
<ul><br />
<li>Needed more phsABC, so ran five PCR reactions to amplify it from plasmid pSB74. Reaction mixture contents were the same as the DMSO variant used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4">6/30 </a>as was the thermocycler protocol (phs50). The results were then run on a gel</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/Yale-pcr-gel.jpg" /><br />
</div><br />
<div id="caption">Gel of PCR amplified phsABC Extreme right lane:1 kb ladder, all other lanes PCR reaction mixtures and their spillover</div><br />
<br />
<li>Needed chloramphenicol plates for pSB1C3-based constructs, so mixed 350 μL of 50 μg/μL chloramphenicol with an autoclaved solution of 7.5 g agar and 12.5 g LB in 500 mL H2O and poured plates. </li><br />
<br />
<b>Digestions</b> <br/><br />
<br />
<li>Ran all three digestion reactions for 30 minutes at 37 °C before heat killing for 20 minutes at 80 °C. Digestion mixtures had the following components: </li><br />
<table><br />
<tr> <td><br />
Digestion Reaction Components<br />
</td></tr> <tr> <td><br />
phsABC Digestion </td> <td> B0015 Digestion </td> <td>pSB1C3 Digestionhttps://2010.igem.org/Main_Page<br />
</td></tr> <tr> <td><br />
5 μL EcoRI Buffer 10x </td> <td>5 μL NEB Buffer 3 10x </td> <td> 5 μL EcoRI Buffer 10x<br />
</td></tr> <tr> <td><br />
0.5 μL BSA 100x </td> <td>0.5 μL BSA 100x </td> <td>0.5 μL BSA 100x <br />
</td> </tr> <tr> <td><br />
21.1 μL phsABC soln (1 μg DNA)</td> <td> 3.8 μL B0015 soln (1 μg DNA) </td> <td>15 μL phsABC soln (375 ng DNA)<br />
</td></tr> <tr> <td><br />
1.8 μL EcoRI & 1.8 μL SpeI </td> <td>1.8 μL XbaI & 1.8 μL PstI </td> <td>1.2 μL EcoRI, 1.2 μL PstI, & 1.2 μL DpnI<br />
</td></tr> <tr> <td><br />
20.8 μL H2O </td> <td>37.1 μL H2O </td> <td> 15.9 μL H2O<br />
</td><br />
</tr><br />
</table><br />
</ul><br />
<i>Wetlab work for this day is also recorded on page 51 of the hard copy lab notebook. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/8--Further ligation efforts<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<br />
<!------------- Thursday -------------><br />
<h4> Ligation Efforts Continue</h4><br />
<ul><br />
<br />
<li>In addition to trying the triple-ligation strategy started on 7/7 (ligation attemp #3) will try trouble-shooting the ligation strategy used in attempts #1 and #2. In particular, will modify digestion protocol and test EcoRI and XbaI (older enzymes) for activity. </li><br />
</ul><br />
<b>Ligation Attempt #3 (Triple Ligation)</b> <br/><br />
<ul><br />
<li>Ran triple ligation all day at room temperature and stored at 4°C. Ligation contents were as follow to have a 1:1:1 stoichiometric ratio of all three components.</li><br />
<table><br />
<tr> <td><br />
Component </td> <td> Volume<br />
</td> </tr><br />
<tr> <td><br />
Distilled Water </td> <td>6.4 μL<br />
</td> </tr><br />
<tr> <td><br />
T4 Ligase buffer 10x </td> <td>2 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB T4 Ligase</td> <td> 1 μL<br />
</td> </tr><br />
<tr> <td><br />
pSB1C3 solution </td> <td> 2 μL<br />
</td> </tr><br />
<tr> <td><br />
B0015 solution</td> <td> 2.4 μL<br />
</td> </tr><br />
<tr> <td><br />
phsABC solution </td> <td> 6.2 μL<br />
</td> </tr><br />
<tr> <td><br />
Total </td> <td> 20 μL<br />
</td> </tr><br />
</table><br />
</ul><br />
<b>Ligation Attempt #4</b><br/><br />
<ul><br />
<li>Concerns about enzyme activity led to reversing the order of the serial digestion of B0015 background so that the first digestion is XbaI (low salt buffer) and the second is EcoRI (high salt buffer). Will also omit the pre-ligation PCR purification step.</li><br />
<li>Overnight digestion of ligation components--Set up the following digestions to run overnight at 37°C. The EcoRI was newly purchased as there was concern about the age of the previously used enzyme.</li><br />
<br />
<table><br />
<tr> <td><br />
<b>phsABC double digestion</b><br />
</td></tr><br />
<tr> <td> <br />
NEB EcoRI Buffer</td> <td> 5 μL<br />
</td></tr><br />
<tr> <td><br />
BSA 100x </td> <td>0.5 μL<br />
</td></tr><br />
<tr> <td><br />
phsABC solution </td> <td> 30.8 μL (1 μg DNA)<br />
</td></tr><br />
<tr> <td><br />
NEB EcoRI </td> <td>1.8 μL<br />
</td></tr><br />
<tr> <td><br />
NEB SpeI </td> <td>1.8 μL<br />
</td></tr><br />
<tr> <td><br />
Sterile H2O </td> <td> 10.1 μL<br />
</td></tr> </table><br />
<table> <tr> <td><br />
<b>B0015 digestion </b> <br />
</td></tr><br />
<tr> <td> <br />
NEB Buffer 4 </td> <td> 5 μL<br />
</td></tr><br />
<tr> <td><br />
BSA 100x </td> <td> 0.5 μL<br />
</td></tr><br />
<tr> <td><br />
NEB XbaI </td> <td> 3.6 μL<br />
</td></tr><br />
<tr> <td><br />
B0015 solution </td> <td> 3.8 μL ( 1 μg DNA)<br />
</td></tr><br />
<tr> <td><br />
Sterile H2O </td> <td>37.1 μL<br />
</td></tr><br />
</table><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 52 and 53 of the hard copy lab notebook.</i> <br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/9--Ligation attempt #4 to join phsABC and B0015 terminator.<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4> Ligation Attempt #4</h4><br />
<ul><br />
<li>Removed both overnight digestions, the XbaI digestion of B0015 and the EcoRI and SpeI digestion of phsABC, from 7/8 from the incubator.</li><br />
<li>Heatkilled enzymes of phsABC digest with 20 minutes at 80°C.</li><br />
<li>Ran <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>on XbaI digestion using microcentrifuge. In order to give a concentrated sample, eluted in only 35 μL of EB Buffer that had been heated at 50°C. Resulting concentration could not be determined as there was residual ethanol contamination.</li><br />
<li>Ran EcoRI digestion of purified XbaI digest product at 37 °C, with components as follows. Digestion was run for an hour before addition of CIP, after which it was run for another hour. </li><br />
<table> <tr> <td><br />
<b>B0015 digestion with EcoRI</b><br />
</td> </tr><br />
<tr> <td> <br />
NEB EcoRI buffer </td><td> 5 μL<br />
</td> </tr><br />
<tr> <td><br />
BSA 100x </td><td> 0.5 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB EcoRI </td><td>3.6 μL<br />
</td> </tr><br />
<tr> <td><br />
B0015 digested with XbaI </td><td>25 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB Calf Intestinal Phosphatase </td><td>1 μL<br />
</td> </tr><br />
<tr> <td><br />
Sterile H2O </td><td> 15.9 μL<br />
</td> </tr><br />
</table><br />
<br />
Followed with 20 minute heatkill at 80°C.<br/><br />
<br />
<li>In order to set up ligation, need concentration of B0015, but ethanol contaminant continues to preclude measurement. Will estimate that the purification between digestion steps resulted in a 50% loss of DNA, leaving behind a 10 ng/μL concentration. Will also, as usual, run different ligation trials with different ratios of insert to vector, which should help combat any issues if this estimate is inaccurate </li><br />
<li>Set up the following ligation trials, with stoichiometric ratios of insert to vector (I:V) based on B0015 concentration estimate. Ran the ligations for 20 minutes at room temperature. </li><br />
<table><br />
<tr><td><br />
<b>Ligation Reaction Components</b><br />
</td></tr><br />
<tr><td><br />
Control Ligation</td> <td> 1:1 I:V </td> <td>2:1 I:V </td> <td> 3:1 I:V</td> <td> 4:1 I:V <br />
</td></tr><br />
<tr><td><br />
2 μL NEB T4 ligase buffer for all reactions<br />
</td></tr><br />
<tr><td><br />
1 μL NEB T4 ligase for all reactions<br />
</td></tr><br />
<tr><td><br />
5 μL B0015 solution for all reactions<br />
</td></tr><br />
<tr><td><br />
12 μL H<sub>2</sub>O </td> <td> 8.9 μL </td> <td>5.8 μL H<sub>2</sub>O </td> <td>2.7 μL H<sub>2</sub>O </td> <td> --<br />
</td></tr><br />
<tr><td><br />
-- </td> <td>3.1 μL phsABC </td> <td> 6.2μL phsABC </td> <td> 9.3 μL phsABC</td> <td> 12 μL phsABC<br />
</td> </tr><br />
</table><br />
<li> Following ligation, transformed 1 μL of each ligation reaction into commercial grade cells according to standard transformation protocol. Also transformed triple ligation reaction. </li><br />
</ul><br />
<br />
<h4>Enzyme Activity Test</h4><br />
<br />
To test enzyme activity, will do single digests of B0015 with enzymes in question and then run them on a gel to see if the plasmid linearized. XbaI and EcoRI are old, so will test them first. For XbaI activity test, simply set aside 10 μL of digestion solution from overnight digestion. Testing of EcoRI required additional digests. Digests using both old and new EcoRI samples were run for two hours at 37 °C with the following components:<br/><br />
<table><br />
<tr><td><br />
<b>EcoRI Activity </b><br />
</td> </tr><br />
<tr><td><br />
NEB EcoRI buffer </td> <td> 5 μL<br />
</td> </tr><br />
<tr><td><br />
BSA 100x </td> <td>0.5 μL<br />
</td> </tr><br />
<tr><td><br />
NEB EcoRI </td> <td>3.6 μL<br />
</td> </tr><br />
<tr><td><br />
undigested B0015 </td> <td>3.8 μL ( 1 μg DNA)<br />
</td> </tr><br />
<tr><td><br />
Sterile H<sub>2</sub>O </td> <td>37.1 μL<br />
</td> </tr><br />
</table><br />
<br />
Followed with 20 minute heatkill at 80°C.<br/><br />
<ul><br />
<li>Loaded all three enzyme activity tests on a 1.0% agarose gel and ran them at 90 V versus a 1 kb ladder and circular B0015 plasmid. </li><br />
<br />
Gel of single digests of B0015 Left to Right lane 1:1 kb ladder, lane 2: uncut circular B0015, its dimer, and lane 3 spillover, lane 3: XbaI digest of B0015, lane 4: old EcoRI digest of B0015, lane 5: new EcoRI digest of B0015<br/><br />
<br />
<li>Based on the results, all enzymes appeared to work, as they successfully created a linearized 3.2 kb fragment.</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/9/93/Yale-enzyme-test.jpg" /><br />
</div><br />
<br />
<i>Wetlab work for this day is also recorded on pages 54-57 of the hard copy lab notebook. </i><br />
</ul><br />
<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/10--Transformants from ligation attempts<br />
<br />
</li><br />
<br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<b> Transformants from ligation attempts #3 an #4</b> <br/><br />
<ul><br />
<li> The plate from ligation attempt #3 shoed in excess of 100 differnt colonies. </li><br />
<li> Plates from ligation attempt #4 had the following colony counts:</li><br />
Control-- 5 colonies <br/><br />
1:1--28 colonies <br/><br />
2:1--38 colonies <br/><br />
3:1--18 colonies <br/><br />
4:1--27 colonies <br/><br />
<li>Took colonies from various plates and used them to establish index plates on Amp LB, labeled as follow: eight colonies from triple ligation as #1-#8, four colonies from 1:1 as#9-#12, four colonies from 2:1 as #13-#16, four colonies from 3:1 as #17-#20, and four colonies from 4:1 as #21-#24.</li><br />
<i>Wetlab work for this day is also recorded on page 58 of the hard copy lab notebook.</i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/11--Prep for copper growth assays of transformants<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
<b>Ligation work </b> <br/><br />
<ul><br />
<li> Realize that mistakenly plated triple ligation reaction on ampicillin plate rather than the required chloramphenicol, so none of the colonies present are of value. Fortunately still have transformation solution, so replate it, this time on a chloramphenicol </li><br />
<li> Inoculated overnight liquid cultures of ligation attempt #4 transformants (#9-#24 on index plates) for miniprep the following morning </li><br />
<b>Growth assay prep work </b> <br/><br />
<li> Want to see if pSB74 confers any copper resistance on <i>E. coli</i>, since it should help them precipitate nearby copper and thus reduce the copper concentration that they experience. </li><br />
<li> Inoculated liquid cultures of LE392 with pSB74 in ampicillin LB and untransformed LE392 in plain LB and left to grow overnight on shaker at 37˚C for growth assays the following day </li><br />
<li> Prepared CuSO<sub>4</sub> solutions in LB. Each well in the 96-well plate will have 225 uL of copper solution and 25 uL of cell solution, so the copper solution must have a CuSO<sub>4</sub> concentration that is 10/9 the desired final values. The copper solutions must also contain ampicillin and IPTG at the same levels as the cultures that will be introduced. The desired CuSO<sub>4</sub> concentrations are those used in the wide and narrow concentration range trials on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> 6/10 </a> and <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_2"> 6/14 </a>. </li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59-61 of the hard copy lab notebook.</i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
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</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4Team:Yale/Our Project/Notebook/Week 42010-10-28T02:50:45Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 4 (6/28 -7/4)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<b> pSB74 transformants</b> <br/><br />
Extensive colony growth was seen on plated transformations of pSB74 into DH5alpha and LE392 from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_3"> 6/25 </a> but none was visible on BL21 plate. Continued failure of BL21 transformation suggests that the homemade competent BL21 cells being used are somehow faulty. <br/><br />
<h4> PCR amplification of phsABC </h4><br />
<ul><br />
<li> Prior to PCR, took aliquots of all primer samples and diluted them first to 100 uM as detailed below by the chart and then again to 10 uM </li><br />
<table><br />
<tr><br />
<td>Primer</td> <td> phsABC_F </td> <td> phsABC_R </td> <td> phsAB_R </td> <td> phsC_F </td><br />
</tr><br />
<tr><br />
<td> Initial Amount </td> <td>38.56 nm</td> <td>46.58 nm</td><td>29.03 nm</td><td>21.94 nm</td><br />
</tr><br />
<tr><br />
<td> Volume of water </td> <td>385.6 uL</td> <td>465.8 uL</td><td>290.3 uL</td><td>219.4 uL</td><br />
</tr><br />
</table><br />
<li> Similarly, diluted the DNA sample to 10 ng DNA/uL. Started with 0.5 uL of miniprepped pSB74 (sample a) at 67.3 ng/uL and added 2.865 uL of water to get the desired concentration.</li><br />
<b> PCR reaction </b> <br/><br />
<li> Added the following to each of three PCR tubes: 10 uL 5x Phusion HF buffer, 27.5 uL water, and 1 uL of dNTPs. 5 uL were added of both the forward and reverse primers (at 10 uM), as was 1 uL of the template DNA. The matching of tube numbers, template DNA, and primers was as follows:</li><br />
<table><br />
<tr><br />
<td> Tube #</td> <td>2</td> <td>3</td> <td> 4</td><br />
</tr><br />
<tr><br />
<td> Template DNA</td> <td>phsC</td> <td>phsAB</td> <td> phsABC</td><br />
</tr><br />
<tr><br />
<td>Primers</td> <td>phsC_F & phsC_R</td> <td>phsABC_F & phsAB_R</td> <td> phsABC_F & phsC_R</td><br />
</tr><br />
</table><br />
<li> Chilled tubes on ice, then added 0.5 uL of Phusion DNA polymerase and put in thermocycler. </li><br />
<li> Prior to designing thermocycler protocol, used oligocalc to get the following Tm values for the primers. </li><br />
<table><br />
<tr><br />
<td> Primer </td> <td>phsABC_R </td><td>phsABC_F </td><td>phsAB_R </td><td>phsC_F </td><br />
</tr><br />
<tr><br />
<td> Tm </td> <td>73˚C </td><td>66˚C</td><td>64˚C</td><td>72˚C </td><br />
</tr><br />
</table><br />
However, these values seem excessively high, so will just start out with an annealing temperature of 55˚C. Devised the following "phs" Thermocycler Protocol<br/><br />
1. Initial Denaturation-- 2 minutes at 98˚C<br/><br />
2. Denaturation--15 seconds at 98˚C<br/><br />
3. Annealing--15 seconds at 55˚C<br/><br />
4. Extension--3 minutes at 72˚C <br/><br />
Steps 2-4 are repeated for 25 cycles<br />
5. Final Extension--10 minutes at 72˚C <br/><br />
6. Hold--indefinitely at 4˚C<br/><br />
<b> Gel of PCR Products </b> <br/><br />
Ran PCR products on an 0.8% agarose gel with 10 uL of ethidium bromide. Loaded 1 uL of each sample with 9 uL of water and 2 uL of loading buffer. Ran gel until done at 60 V, but had difficulty imaging so left aside to deal visualize the next day, unaware that it would spread. <br/><br />
</ul><br />
<i>This day's labwork is also recorded on pages 23-28 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 6/29--Gel extraction of PCR amplified phs, TSI agar slant assay for hydrogen sulfide production, & pre-ligation double digestion<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>PCR amplification of phs </h4> <br />
<li> Because the gel run the previous day was no longer good, had to run a second 0.8% agarose gel of the PCR products to determine the success of the PCR reaction. Below is the image of the gel, with a 1 kb ladder in the leftmost lane, phsC in next lane, phsAB in lane 3, and phsABC in lane 4. </li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/68/Yale-1st-phs-pcr.jpg" /><br />
</div><br />
<div id="caption"><br />
The phsC fragment seems to have been amplified with decent success, but phsABC and phAB <br /> are only present in trace amounts, so the thermocycler protocol will have to be tweaked. </div> <br /><br />
<li> In the mean time, gel extract the phsC from lane 2 and the phsABC from lane 4 using the microcentrifuge variant of the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol </a> </li><br />
<br />
<h4> TSI Agar Slant Assay </h4><br />
<li> Want to confirm that IPTG will induce H<sub>2</sub>S production in pSB74 transformants, so inoculate 5 mL Amp/LB liquid cultures of DH5alpha and LE392 transformed with pSB74 and grow them up on shaker at 37˚C for use in TSI agar assay. </li><br />
<li> The slants don't contain IPTG, so must inject IPTG with the transformants into the slants. Took 200 uL of each cell solution and added to it 50 uL of 1 M IPTG, then injected the resulting mixture into a slant. Left the slants to sit overnight in the fume hood </li><br />
<br />
<h4> Double digestion in preparation for ligation </h4><br />
In order to create the desired Biobricks, must insert the phsABC operon into terminator B0015 and put the constitutive promoter J23114 into the generator P0312. Following the Biobrick combination protocol, we then must digest phsABC and J23114 with EcoRI and SpeI and digest B0015 and P0312 with EcoRI and XbaI. The former can be done as a double digest, but the latter must be done sequentially because the enzymes require different buffers.<br/><br />
Set up the following 50 uL digestions:<br/><br />
<b>Digest of phsABC</b>--5 uL EcoRI buffer, 0.5 uL 100x BSA, 40 uL phsABC fragment (from gel extraction), 1.8 uL EcoRI, 1.8 uL SpeI, 0.9 uL water <br/><br />
<b>Digest of J23114</b>--5 uL EcoRI buffer, 0.5 uL 100x BSA, 8 uL miniprepped J23114 (1 ug DNA), 1.8 uL EcoRI , 1.8 uL SpeI, 32.9 uL water <br/><br />
<b>Digest of B0015</b>--5 uL EcoRI buffer, 12 uL miniprepped B0015 (1 ug DNA), 3.6 uL EcoRI , 29.4 uL water <br/><br />
<b>Digest of P0312</b>--5 uL EcoRI buffer, 9 uL miniprepped P0312 (1 ug DNA), 3.6 uL EcoRI , 32.4 uL water <br/><br/><br />
Let each digestion run for two hours at 37˚C, then ran the result through the microcentrifuge version of the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol.</a><br/><br />
<br />
<br />
<i>This day's labwork is also recorded on pages 28-31 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 6/30--Troubleshooting PCR amplification of thiosulfate reductase gene & continued work toward ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>TSI Agar slant </b></br><br />
<ul><br />
<li>No visible color change, and decide that it is safe to accelerate growth by moving slants to the 37˚C incubator as research fails to turn up any safety concerns regarding the hydrogen sulfide emitted in this kind of assay. </li><br />
</ul><br />
<b>PCR efforts continue </b><br/><br />
<ul><br />
<li>Redid 6/29 PCR of phsABC and phsAB, but this time ran two trials of each, with and without 3% DMSO, and changed the thermocycler protocol so that the annealing temperature was 50˚C rather than 55˚C, calling the new protocol "phs50."</li><br />
<li> After running the protocol, found that PCR tubes had popped open and contents had evaporated, so set up PCR a second time to run overnight.</li><br />
</ul><br />
<h4>Vector digestion & 1st ligation attempt</h4><br />
<ul><br />
<li> Ran the second portion of the B0015 and P0312 sequential digests, with reaction contents of each as follows: 5 uL NEB Buffer 4, 0.5 uL 100x BSA, 3.6 uL XbaI, 40 uL DNA from PCR purification protocol, and 0.9 uL water. Let the reactions run for 2.5 hours at 37˚, but after 1.5 hours added to each reaction 1 uL of Calf Intestinal Phosphatase (CIP) to cleave terminal phosphates and discourage self-ligation in the next step. </li><br />
<b>Ligation of phsABC and B0015</b> <br/><br />
By this point the DNA concentrations of the digestion reactions are dismally low (around 2 ng/uL for B0015 and phsABC), but will try a ligation anyways while at the same time preparing for a possible redo. Set up digestion reactions of two sizes, 10 uL and 20 uL, with a control reaction for each in which there is no insert.<br/><br />
<b> Reaction 1--small control</b> 1 uL ligase buffer, 0.5 uL T4 ligase, 4.25 uL B0015, 4.25 uL water<br/><br />
<b> Reaction 2--small 1:1 insert:vector</b>1 uL ligase buffer, 0.5 uL T4 ligase, 4.25 uL B0015, 4.25 uL phsABC<br/><br />
<b> Reaction 3--small 2:1 insert:vector</b>1 uL ligase buffer, 0.5 uL T4 ligase, 2.5 uL B0015, 6 uL phsABC<br/><br />
<b> Reaction 4--large control</b>2 uL ligase buffer, 1 uL T4 ligase, 8.5 uL B0015, 8.5 uL water<br/><br />
<b> Reaction 5--large 1:1 insert:vector</b>2 uL ligase buffer, 1 uL T4 ligase, 8.5 uL B0015, 8.5 uL phsABC<br/><br />
Insert to vector ratios are rough and based on the fact that the two are at similar concentrations and are of similar sizes. <br/><br />
Each reaction was let to run 20 minutes at room temperature before being transformed into Xl1-Blue cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a> and then plated on ampicillin LB plates and let to grow overnight.<br/><br />
</ul><br />
<b> Cultures for miniprep & glycerol stock redo </b> <br/><br />
In preparation for potential ligation failure, once again inoculated liquid cultures of Amp LB for miniprep with pSB74,P0312,R0011, B0015, and J23114 transformants, making two 5 mL cultures for each plasmid. When OD reached 0.6, once again made glycerol stocks from 0.5 mL of each solution and 0.5 mL of filter-sterilized 50% glycerol. This time flash-froze the stocks immediately, rather than waiting overnight as on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_3">6/23</a> <br/><br />
<br />
<br />
<i> This day's labwork is also recorded on pages 32-36 of the hard copy lab notebook. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/1--Smelly bacteria (yay!) & ongoing ligation efforts<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4> TSI agar assay </h4><br />
<ul><br />
<li>LE392 and DH5alpha cultures in TSI agar showed some growth. No black color change was visible, but both slants had a rotten egg scent, so suspected generation of H<sub>2</sub>S at concentrations too low to visibly trigger color change yet.</li><br/><br />
</ul><br />
<h4>PCR work</h4><br />
<ul><br />
<li>Ran 1.0% agarose gel of four 6/30 PCR products at 90 V.</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/87/Yale-phs-gel.jpg" /><br />
</div><br />
<br /><br />
<div id="caption"><br />
Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO<br />
Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO<br />
</div><br />
<br /><br />
<li>Based on gel, it appears that the phs50 thermocycler protocol is an improvement from phs protocol and that including DMSO also improves yield</li><br />
<li>Cut out largest band for each lane and ran through <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol.</a></li> <br />
</ul><br />
<br />
<b>Stockpiling plasmids for later</b> <br/><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">Miniprepped </a> LE392 cultures grown overnight,resulting in two Eppendorfs apiece of the following plasmids: B0015 (double terminator), J23114 (constitutive promoter), pSB74 (thiosulfate reductase), R0011 (IPTG-inducible promoter), and P0312 (lacI needed with R0011). Nanodropping gave the following concentrations:</li><br/><br />
<table><br />
<tr><br />
<td> Sample </td><td> B0015-1</td><td> B0015-2 </td><td>P0312-1</td><td> P0312-1</td><td> J23114-1 </td><td> J23114-2</td><td> pSB74-1</td><td> pSB74-2 </td><td> R0011-1 </td><td> R0011-2 </td><br />
</tr><br />
<tr><br />
<td> Concentration (ng DNA/uL) </td><td> 262.3</td><td> 28569</td><td>129.1</td><td> 92.3</td><td> 189.0 </td><td> 202.6</td><td> 214.4</td><td> 284.4 </td><td> 102.6</td><td> 79.6 </td><br />
</tr><br />
</table><br />
</ul><br />
<h4>Results of 6/30 Ligation (Ligation Attempt #1)</h4><br />
<ul><br />
<li>Plated post-ligation transformants showed the following results: </li><br />
<br />
Reaction #1 (small control) 0 colonies<br/><br />
Reaction #2 (1:1 insert:vector) 2 colonies, numbered 1A and 2A<br/><br />
Reaction #3 (2:1 insert:vector) 0 colonies<br/><br />
Reaction #4 (large control) 2 colonies<br/><br />
Reaction #5 (large 1:1 insert vector) 2 colonies, numbered 3A and 4A<br/><br />
<br />
Concerned about the small number of colonies and low ratio of number of colonies from actual ligation to number of colonies from control ligation.<br/><br />
<br />
<li>Started liquid cultures and index plates of four colonies from reactions 2 and 5.</li><br />
<li>Also set up colony PCR for all four colonies, spotting the template DNA into tubes with the following contents and running on thermocycler protocol phs50 </li> <br />
<table><br />
<tr><br />
<td>Component</td> <td>Volume</td><br />
</tr><br />
<tr><br />
<td>Distilled Water</td> <td>27 μL</td><br />
</tr><br />
<tr><br />
<td>Phusion 5x Buffer</td> <td>10 μL</td><br />
</tr><br />
<tr><br />
<td>DMSO</td> <td>1.5 μL</td><br />
</tr><br />
<tr><br />
<td>10 mM dNTPs </td> <td>1 μL</td><br />
</tr><br />
<tr><br />
<td>10 uM phsABC_F primer</td> <td>5 μL</td><br />
</tr><br />
<tr><br />
<td>10 uM phsABC_R primer </td> <td>5 μL</td><br />
</tr><br />
<tr><br />
<td>Phusion Polymerase</td> <td>0.5 μL</td><br />
</tr><br />
<tr><br />
<td>Template DNA </td> <td>spotting</td><br />
</tr><br />
<tr><br />
<td>Total</td> <td>50 μL</td><br />
</tr><br />
</table><br />
<li>After PCR, ran 1.0% agarose gel of colony PCR reaction solutions at 90 V versus a 1 kb ladder. A preliminary visualization showed no visible non-primer DNA except for a band between 3 and 4 kb for the PCR product from colony 1. Based on a later visualization said band appeared to be closer to 3 kb, suggesting the 3.2 kb B0015 vector rather than the 3.6 kb insert. Unfortunately gel was dropped and damaged irreparably before picture could actually be taken. </li><br />
</ul><br />
<br />
<i>Wetlab work for this day is also described on pages 37-41 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/2--Confirmation of hydrogen sulfide production plus more ligation work<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4> TSI Agar slants </h4><br />
<ul><br />
<li>Slant inoculated with the LE392 transformants containing pSB74 showed a mass of black precipitate near the surface, definitively confirming the production of hydrogen sulfide (!), the smell of which was by now very strong. The DH5alpha transformants did not exhibit the same H<sub>2</sub>S odor.</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/3/38/Yale-tsi-agar.png" /><br />
</div><br />
<div id="caption">blurry (apologies!) image of LE392 slant showing FeS precipitate(right) next to un-inoculated slant (left)</div><br />
<br />
</ul><br />
<h4>Ligation Attempt #2</h4><br />
<ul><br />
<li>Still don't know whether ligation attempt #1 was successful, as colony PCR on 7/1 was not definitive. Will sequence the results for a certain answer, but given the small number of colonies think it best to redo ligation in the interim in case of failure.</li><br />
<li>To this effect redid double digestion of phsABC insert with EcoRI and SpeI and the serial digestion of B0015 with EcoRI and then XbaI. Simultaneously digested J23114 to be inserted into P0312, which lacks a promoter. Each digestion reaction had a total volume of 100 uL and included 2 ng of DNA, double the amount used previously, so that even after purification there would still be a sizable amount of DNA remaining for ligation. Otherwise digestion and purification was run the same as on 6/29 & 6/30. </li><br />
</ul><br />
<i>This day's activities are also recorded on pages 42-45 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/3--More attempts to put a terminator on thiosulfate reductase and a promoter on lacI<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Ligation Attempt #2 continued</h4><br />
<ul><br />
<li>Following digestion and purification, took concentrations of digestion products, which were still miserably low (phsABC: 3.2 ng/uL, P0312: 16.88 ng/uL, J23114: 23.98 ng/uL, B0015: 13.24 ng/uL) and set-up ligation attempt #2. For each of the two ligations (phsABC into B0015 and J23114 into P0312) four different 20 uL reactions were run: a control ligation with vector, ligase and buffer but no insert, a reaction with a 2:1 stoichiometric ratio of insert to vector, a reaction with a 3:1 insert to vector ratio, and a reaction with a 5:1 insert to vector ratio. All reactions used 1 uL NEB T4 ligase and 2 uL of accompanying 10x buffer and were run for 15 minutes at room temperature. Following this the (theoretically) ligated plasmids were transformed into competent XL1-Blue cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard transformation protocol </a> and plated onto Amp plates</li><br />
<li>Index plates from ligation attempt #1 on 7/1 belatedly showed growth. </li><br />
</ul><br />
<i>This day's activities are also recorded on page 45 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/4--Ligation attempt #2 transformants<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
<h4> Transformed Colonies from Ligation Attempt #2</h4><br />
<ul><br />
<br />
<li>Checked on plated transformants and observed the following: </li><br />
<br />
<b>phsABC/B0015 system</b><br/> <br />
Reaction #1 (control) 2 colonies<br/> <br />
Reaction #2 (2:1 insert:vector) 4 colonies, numbered 1B-4B<br/><br />
Reaction #3 (2:1 insert:vector) 5 colonies, numbered 5B-9B<br/><br />
Reaction #4 (large control) 0 colonies<br/><br />
<b>J23114/P0312 system </b> <br/><br />
Reaction #5 (control) numerous* colonies <br/><br />
Reaction #6 (2:1 insert:vector) numerous colonies, somewhat more than #5<br/><br />
Reaction #7 (3:1 insert:vector) numerous colonies,about double #5<br/><br />
Reaction #8 (5:1 insert:vector) numerous colonies, about double #5<br/><br />
<br />
numerous=more than readily countable<br/><br />
<br />
<li>Inoculated 5 mL liquid Amp/LB cultures with colonies 1B-9B and let grow overnight at 37 on shaker </li><br />
<li> Set aside plates 6-8 in the fridge to work with later. </li><br />
</ul><br />
<br />
<i>This day's activities are also recorded on page 46 in the hard copy of the lab notebook. </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
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</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_3Team:Yale/Our Project/Notebook/Week 32010-10-28T02:50:18Z<p>Litagaki: </p>
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<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 3 (6/21-6/27)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK: NEEDS TO BE EDITED-------------><br />
<br />
<li><br />
<br />
Monday 6/21--Miniprep Biobrick plasmids from overnight culture, but a mix up samples, necessitating a diagnostic digest.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<li>Miniprepped the cultures grown overnight according to <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> standard microcentrifuge protocol, </a> but mixed up tubes so hope to sort them out with a diagnostic double digest</li><br />
<li> The similar size and identical backgrounds of the Biobricks makes many of them difficult to distinguish by restriction digest failing cut sites within the Biobricks themselves. EcoRI and PstI are available but B0015, C0012, and J23114 (all 35 bp in around 3 kb backgrounds) are too hard to tell apart by such a digestion. However, a EcoRI/PstI digest should be able to tell them from R0011 and pSB74--the former will be divided into 1.1 kb and 2.1 kb fragments while the latter will end up as 3.6 kb and 9.4 kb fragments.</li><br />
<li> Before setting up digestion, used nanodrop to determine the concentration of the ten unknown samples (two of each plasmid) </li><br />
<table><br />
<tr><br />
<td>Sample</td><br />
<td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><br />
</tr><br />
<tr><br />
<td>DNA Concentration (ng/ul)</td><br />
<td> 22.0 </td><td> 76.1 </td><td>51.5</td><td>15.5</td><td>98.7</td><td>303.0</td><td>27.9</td><td>11.4</td><td>82.6</td><td>306.6</td><br />
</tr><br />
</table><br />
<li>Samples 2,4, and 7 were too dilute to be of use, but ran 50 uL digestions of the other seven using 1 ng worth of DNA,0.5 uL of 100x BSA, 5 uL of NEB EcoRI 10xBuffer, 0.5 uL of NEB EcoRI, 0.5 uL of NEB PstI, and enough water to round out the volume. These digestions were put in the thermocycler for 30 minutes at 37˚C </li><br />
<li> Ran 10 uL of each digestion reaction with 2 uL loading buffer versus a 1 kb ladder in a 0.8% agarose gel stained with EtBr. The resulting image is shown below, with columns containing the ladder, sample 2, sample 3, sample 5, sample 6, sample 8, sample 9, and sample 10 from left to right. </li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/4/40/Yale-plasmid-check.jpg" /><br />
</div><br />
<br /><br />
<li> Samples 3 and 10 match the fragment sizes expected for R0011, while 5 might be pSB74, though the fragment size seems rather large. The other assignments are rather ambiguous, so gave up on identifying tubes. </li><br />
<li> Redid inoculation of liquid cultures of strains containing pSB74, R0011, J23114, C0012, and B0015 and left in ampicillin LB on the shaker overnight. </li><br />
</ul><br />
<i> This day's activities are also recorded on pages 18 and 19 of the hard copy lab notebook. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 6/22--Miniprep redos<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<ul><br />
<li>Began miniprep of overnight cultures, but in initial cell pelleting phase the centrifuge spun the tops of the tubes, spilling them. </li><br />
<li> Started over with the inoculation of two 5 mL liquid ampicillin LB cultures each of pSB74, R0011, C0012,J23114, and B0015 containing strains. </li><br />
<li> When OD was approximately 0.6, took 0.5 mL of each solution and mixed with 0.5 mL of 50% filter-sterilized glycerol and set aside for glycerol stocks. </li><br />
<li> Let cultures continue to grow all day, then miniprepped via the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> microcentrifuge protocol, </a> eluting into 40 uL of water. </li><br />
</ul><br />
<i> This day's activities are also recorded on page 18 of the hard copy lab notebook. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
Wednesday 6/23--Making TSI agar slants & transformation of P0312<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Nanodrop of 6/22 miniprep </b> <br/><br />
Nanodropped the ten minipreps from the day before and got the following results:<br />
<table><br />
<tr><br />
<td>Sample</td><br />
<td>J23114a</td><td>J23114b</td><td>C0012a</td><td>C0012b</td><td>R0011a</td><td>R0011b</td><td>B0015a</td><td>B0015b</td><td>pSB74a</td><td>pSB74b</td><br />
</tr><br />
<tr><br />
<td>DNA Concentration (ng/ul)</td><br />
<td> 122.9</td><td> 91.3 </td><td>17.4</td><td>57.9/td><td>86.8</td><td>59.5</td><td>70.2</td><td>83.7</td><td>67.3</td><td>44.2</td><br />
</tr><br />
</table><br />
Also flash froze in liquid N<sub>2</sub> the glycerol DNA solutions from 6/22 and put them in the -80˚C freezer. <br/><br />
<b> Triple Sugar Iron (TSI) agar preparation</b> <br/><br />
So that can readily determine whether bacteria are producing hydrogen sulfide, created a batch of TSI agar slants according to the following <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/TSI_agar"> recipe </a>, with ampicillin as the added antibiotic and tubes of the medium allowed to solidify on an angle to produce a characteristic slant. If a culture containing thiosulfate reductase is grown in such a slant, it will turn the medium black by precipitating out black iron sulfide, an obvious visual cue of hydrogen sulfide production. <br/><br />
<b> Transformation of BBa_P0312</b> <br/><br />
After realizing that Biobrick P0312 can be used in place of C0012 and R0011, transformed LE392 with P0312 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and plated on ampicillin LB plates to grow overnight in the incubator at 37˚C. (Because no Amp LB plates were prepared, simply spread the antibiotic topically on a plain LB plate prior to adding transformants.)<br/><br />
<i> This day's labwork is also recorded on pages 18, 20, and 21 of the hard copy lab notebook. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<li><br />
<br />
Thursday 6/24--primer ordering & IPTG-inducible promoter(P0312) transformants<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<ul><br />
<li>Finally ordered the primers designed on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_2"> 6/15 </a> as well as standard VF2 and VR primers for Biobricks. </li><br />
<li> Saw that P0312 transformants had grown, though apparently ampicillin was not thoroughly distributed on the plate, as there was lawn growth at the very edges and scattered colonies in the middle. Used one of these middle resistant colonies to inoculate a 5 mLliquid ampicillin LB culture to grow overnight on the shaker at 37˚C for miniprep the next day. </li><br />
</ul><br />
<i> This day's labwork is also recorded on page 22 of the hard copy lab notebook. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 6/25--P0312 miniprep, pSB74 transform, & pouring plates<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<ul><br />
<li> Miniprepped the overnight culture of P0312 in LE392 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> standard microcentrifuge protocol </a> and found the resulting plasmid solution to have a concentration of 11.9 ng DNA/uL</li><br />
<li>Transformed BL21, LE392, and DH5alpha with pSB74 according to this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> <br/> and plated on ampicillin LB plates to incubate overnight at 37˚C. </li><br />
<li> Prepared 1 liter's worth of ampicillin LB plates and 500 mL of plain LB plates according to this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/LB_agar"> recipe. </a> The ampicillin 1000x stock solution was made by dissolving 2.0 g of ampicillin in 20.0 mL of water, filter-sterilizing the resultant solution, and storing it in 1 mL aliquots in the freezer. </li?<br />
<li> Incubated five of the plain LB plates overnight as a sterility check. </li><br />
</ul><br />
<i>This day's activities are also recorded on page 22 of the hard copy lab notebook. </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<!------------- LAB NOTEBOOK: NEEDS TO BE EDITED -------------><br />
</ul><br />
</p><br />
</div><br />
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<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_2Team:Yale/Our Project/Notebook/Week 22010-10-28T02:49:32Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 2 (6/14-6/20)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 6/14--Redo of <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook">6/11</a> assay of bacterial growth within a narrow copper(II) concentrations after analysis of data showed uniformly poor growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Work Continues </h4><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8f/Yale-growthfail.jpg" /><br />
</div><br />
<ul><br />
<li>As the growth assay of <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook">6/11</a> showed uniformly poor growth even at the lowest copper concentrations (see Dh5alpha growth above), redid it with care not to let the liquid cultures overgrow, a possible source of the cultures' previous poor performance. (add plot)</li><br />
<li>In redoing plate assay, changed copper concentration line-up slightly, eliminating 1.25 mM and 2.5 mM trials to allow room for the control concentrations, 0 and 1 M, and only worked with high because there was no real distinction in the three different OD trials done on 6/10. For simplicity, future assays will only use one starting OD (0.075) per strain. </li><br />
<li>During the repeat, noticed that DH5alpha liquid culture grown up for plate reader assay grew at a rate much slower than that of LE392, creating a need to keep diluting the LE392 solution with more LB so it would not overgrow before the DH5alpha was ready. In the end, the culture of LE392 used in the assay was diluted from an OD of 0.761 and the DH5alpha culture from an OD of 0.841.</li><br />
</ul><br />
<i> This day's work is also recorded on pages 8-10 of the lab notebook hard copy. </i><br />
<br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 6/15--Results and analysis of the 6/14 narrow concentration range growth assay as well as planning for the creation of a standard iGEM plasmid bearing the thiosulfate reductase operon (phsABC).<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> Results and Conclusions of Narrow Concentration Range Growth Assay of 6/14</h4><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/d/d1/Yale-dh5alpha-narrow.jpg" /><br />
</div><br />
<ul><br />
<li> Negative readings for the 4 mM samples suggest that there was some sort of irregularity with the blank solution, so will disregard those results. </li><br />
<li> Can see growth really starts to slow down at 3 mM concentrations of Cu<sup>2+</sup> </li><br />
<li>It’s unclear whether the cells at the higher copper concentrations die or simply stop growing. Spot a drop from each well of the plate onto a LB agar plate and incubate overnight at 37˚C. Will check for growth to determine survival of different strains at different copper concentrations.</li><br />
</ul><br />
<i>This information is also recorded on page 11 of the hard copy lab notebook. </i> <br/><br />
<h4> Plasmid Ligation Strategy</h4><br />
<b> The Plan for phs plasmid creation </b> <br/><br />
The phs operon has three genes of interest to us, A, B, and C, that code for different protein products and together are responsible for hydrogen sulfide production. We will use these genes to create a number of different plasmids described below. <br/><br />
<b> Plasmids to be made: </b> <br/><br />
Plasmid 1 gives our basic thiosulfate reductase pathway under IPTG control. Plasmid 5 is a light-controlled analog that we will make if we can find a suitable light-inducible promoter. Plasmid 2 is a promoter-less “generator” to be archived so other teams can use it. Plasmids 3 & 4 are a set and together give the entire pathway. The idea is that since protein production takes a while we will make only part of the pathway inducible. The cell will constitutively produce the A & B products, so after the light hits only the small C protein remains to be made. <br/><br />
<table border="1"><br />
<tr><br />
<td>Product</td><br />
<td>Promoter</td><br />
<td>Gene</td><br />
<td>Terminator</td><br />
<td>Plasmid Vector</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>IPTG-inducible BBa_R0011(well 6G plate 1)</td><br />
<td> phsABC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>none)</td><br />
<td> phsABC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>Constitutive BBa_J23114 (well 20I plate 1)</td><br />
<td> phsAB </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1T3 (Tet resistance)</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>Light-inducible</td><br />
<td> phsC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>light-inducible</td><br />
<td> phsABC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
</table> <br />
*This plan was eventually altered, as promoter choices were altered and no light-inducible promoter was found. <br/><br />
<br /><br />
<div id="right"><br />
<img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /><br />
</div><br />
To make these plasmids, we will rely on the standard iGEM assembly protocol involving restriction enzymes EcoRI, XbaI, PstI, and SpeI shown in the diagram at right, but in the first ligation the B0015 terminator will take the place of C0010 and the phsABC gene in pSB74 will take the place of B0034. <br/><br />
<br />
<b> Steps to make these plasmids </b> <br/><br />
1. Initial Amplification—transform and amplify terminator, promoter, and gene DNA <br/><br />
2. Gene-Terminator linkage—Digest terminator with EcoRI and XbaI, digest PCR product gene with EcoRI and SpeI, and ligate together to get gene and terminator in nonstandard plasmid <br/><br />
3. Creation of Standard Generator—digest step 2 product with EcoRI and PstI and ligate into standard iGEM plasmid pSB1C3 to get the generator <br/><br />
4. Addition of promoter—Digest promoter with EcoRI and Spe--digest generator vector with EcoRI and XbaI, and ligate together<br/><br />
<br />
<h4> Primer design for PCR amplification of the phsABC gene from background vector pSB74 </h4><br />
Given the size of phsABC, introducing it in pieces with AB on one plasmid and C on a second might enhance expression. For this purpose, we have designed forward primers that include the EcoRI and XbaI cut sites and an RBS to go at the beginning of A and C as well as reverse primers with a SpeI site to go at the end of B and C. <br/><br />
The following primers were designed based on the phsABC sequence information (see below) after having confirmed that the BioBrick restriction enzyme cut sites are not present in the phsABC sequence. <br/><br />
<br />
N-terminal primer for phsABC: 5’ GCA GAATTC GCGGCCGC T TCTAGA G AGGAGGTTTAT ATG AGC ATT AGT CGT CG <br/><br />
C-terminal primer for phsAB: 5’ AGC ACTAGT A TCA TGC TTT ACC CTC CTG ATG GAC <br/><br />
N-terminal primer for phsC: 5’ GCA GAATTC GCGGCCGC T TCTAGA G AGGAGGTTTAT ATG AAT ACT ATC TGG GG <br/><br />
C-terminal primer for phsABC: 5’ AGC ACTAGT A TCA GAC GGC GGA CTT ATC CCC <br/><br />
<br />
<a id="link" href="javascript:ReverseDisplay('sequence')">See/hide phsABC sequence </a><br />
<div style="display:none;" id="sequence"><br />
<br />
<!------------- sequence data-------------><br />
phsA STM2065 <br/><br />
>sp|P37600|PHSA_SALTY Thiosulfate reductase OS=Salmonella typhimurium GN=phsA PE=3 SV=2 <br/><br />
MSISRRSFLQGVGIGCSACALGAFPPGALARNPIAGINGKTTLTPSLCEMCSFRCPIQAQ VVNNKTVFIQGNPSAPQQGTRICARGGSGVSLVNDPQRIVKPMKRTGPRGDGEWQVISWQ QAYQEIAAKMNAIKAQHGPESVAFSSKSGSLSSHLFHLATAFGSPNTFTHASTCPAGKAI AAKVMMGGDLAMDIANTRYLVSFGHNLYEGIEVADTHELMTAQEKGAKMVSFDPRLSIFS SKADEWHAIRPGGDLAVLLAMCHVMIDEQLYDASFVERYTSGFEQLAQAVKETTPEWAAA QADVPADVIVRVTRELAACAPHAIVSPGHRATFSQEEIDMRRMIFTLNVLLGNIEREGGL YQKKNASVYNKLAGEKVAPTLAKLNIKNMPKPTAQRIDLVAPQFKYIAAGGGVVQSIIDS ALTQKPYPIKAWIMSRHNPFQTVTCRSDLVKTVEQLDLVVSCDVYLSESAAYADYLLPEC TYLERDEEVSDMSGLHPAYALRQQVVEPIGEARPSWQIWKELGEQLGLGQYYPWQDMQTR QLYQLNGDHALAKELRQKGYLEWGVPLLLREPESVRQFTARYPGAIATDSDNTYGEQLRF KSPSGKIELYSATLEELLPGYGVPRVRDFALKKENELYFIQGKVAVHTNGATQYVPLLSE LMWDNAVWVHPQTAQEKGIKTGDEIWLENATGKEKGKALVTPGIRPDTLFVYMGFGAKAG<br />
AKTAATTHGIHCGNLLPHVTSPVSGTVVHTAGVTLSRA <br/><br/><br />
<br />
>phsA <br/><br />
atgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcg ctgggcgcttttccgcccggggcgctggcgcgcaacccgattgccggcattaacggtaaa accacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcag gtagtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacg cgcatatgcgccaggggcgggagcggcgtcagcctggtcaatgacccgcaacggattgta aaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaa caggcttaccaggaaatcgcggcgaaaatgaatgccatcaaagcgcagcatggccccgag agcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacg gcctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccatt gcggcaaaagtgatgatgggcggcgatctggcgatggatatcgctaacacgcgctatctg gtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatg accgcgcaggagaagggggccaaaatggtgagcttcgatccgcgtttgtcgatattttcc agcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcg atgtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttatacc agcggatttgaacagttagcacaggcggtaaaagagacgacgccggaatgggccgccgcg caggccgatgtcccagccgacgttattgtccgggtgacacgcgaactggctgcctgcgcg cctcacgctattgtcagtcctggtcatcgcgcgacgttctcgcaggaagagatcgatatg cggcgtatgatttttacgcttaatgtgctgctcggtaatattgagcgcgaaggcgggcta tatcagaaaaaaaacgcgtctgtttacaataaactggccggagaaaaggtcgcgccaacg ctggcgaaactcaacattaaaaatatgccgaaaccgacggcgcaacgcatcgatttggtc gcaccgcagtttaaatatatcgccgctggcggcggcgtggtgcaaagcattattgactcg gcgttaacccagaagccttacccgataaaggcgtggattatgtcgcggcataatcctttc cagaccgtcacctgtcgttcggacctggtaaaaaccgttgagcaactggatctggtggtc agctgcgatgtctatttgagcgagagcgcggcatatgccgactatctgctgccggaatgc acctatctcgaacgggacgaagaggtatccgatatgtcgggactgcacccggcttacgct ctgcgccaacaggtcgtagagccgattggcgaggcgcgtccgagttggcaaatctggaaa gaacttggcgagcagttgggattagggcagtactatccgtggcaggatatgcagacgcgc caactctatcagttgaacggcgaccatgccttagcgaaggaactgcgacagaaagggtat ctcgaatggggcgttccgctgctattacgcgaaccagaatccgttcgtcagtttacggcg cgttaccccggcgctatcgcgacggacagtgacaacacctatggcgaacagcttcgcttc aaatcgccctccggcaaaatcgaactttattccgcaaccctggaggaattgctccctggc tacggcgttccgcgcgttcgtgactttgcgctgaaaaaagagaatgagctttacttcatt cagggcaaggtggccgtgcataccaatggcgcgacgcagtacgtacctttactcagcgag ctaatgtgggataacgcggtctgggttcatccgcaaacggcgcaagaaaaaggcattaag accggcgatgagatctggctggaaaatgccacgggtaaagagaaaggtaaggcgctggtg acgcccggtatccgcccggacacgctttttgtctatatgggatttggcgctaaagctggg gccaaaacggcggcgacgacacacggtatccactgcggaaatttactgccgcacgtgacg agtccggtatccggtacggtagtgcataccgcaggcgtgacgctgagccgggcatga <br/><br/><br />
<br />
phsB STM2064 <br/><br />
>sp|P0A1I1|PHSB_SALTY Thiosulfate reductase electron transport protein phsB OS=Salmonella typhimurium GN=phsB PE=4 SV=1 <br/> MNHLTNQYVMLHDEKRCIGCQACTVACKVLNDVPEGFSRVQVQIRAPEQASNALTHFQFV RVSCQHCENAPCVSVCPTGASYRDENGIVQVDKSRCIGCDYCVAACPFHVRYLNPQTGVA DKCNFCADTRLAAGQSPACVSVCPTDALKFGRLDESEIQRWVGQKEVYRQQEARSGAVSL <br />
YRRKEVHQEGKA <br/><br/><br />
<br />
>phsB <br/><br />
Atgaatcatttaacgaatcagtacgtcatgctgcatgatgaaaaacgttgtatcggctgc<br />
caggcgtgtaccgttgcctgcaaagtgcttaatgacgtaccggagggatttagccgcgta<br />
caggtacaaattcgcgcccctgaacaggcatccaacgcactaacccattttcaatttgtc<br />
cgcgtctcctgtcagcactgtgaaaatgcgccatgtgtcagcgtttgtcctaccggagcg<br />
tcatatcgtgatgaaaacgggatcgtgcaggtggataaatcgcgctgtattggctgcgat<br />
tattgtgttgccgcgtgtcctttccatgtgcgctatttgaatccgcaaaccggtgtcgcc<br />
gacaagtgtaacttctgcgccgacacgcggttggcggctggccagtctccggcgtgcgta<br />
tccgtttgcccaacggacgcgctgaaattcggcagactggatgagagcgagatccagcgc<br />
tgggtcggtcagaaagaggtctatcgccagcaggaggcgcgtagcggcgcggtcagtctg<br />
taccgtcgtaaagaagtccatcaggagggtaaagcatga <br/><br/><br />
<br />
phsC STM2063 <br/><br />
>sp|P37602|PHSC_SALTY Thiosulfate reductase cytochrome B subunit OS=Salmonella typhimurium GN=phsC PE=4 SV=2 <br/><br />
MNTIWGAELHYAPDYWPLWLIYAGVVVLLMLVGLVIHALLRRMLAPKTAGGEEHRDYLYS LAIRRWHWGNALLFVLLLLSGLFGHFSLGPVALMVQVHTWCGFALLAFWVGFVLINLTTG NGRHYRVNFSGLVTRCIRQTRFYLFGIMKGEAHPFVATEQNKFNPLQQLAYLAIMYALVP LLIITGLLCLYPQVAGLGPVMLVLHMALAIIGLLFICAHLYLCTLGDTPGQIFRSMVDGY <br />
HRHRTAPRGDKSAV <br/><br/><br />
<br />
>phsC <br/><br />
Atgaatactatctggggagcggaactacattatgcgccagattattggccgctgtggttaa<br />
tttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcg<br />
ccggatgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctg<br />
gcgattcgccgctggcattggggaaatgcgttactgtttgttttattactgttaagcggtt<br />
tatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtgg<br />
ttttgccttactggctttctgggtcgggtttgtgctgatcaacctcaccacaggtaacggg<br />
cgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgttttt<br />
acctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagtt<br />
caatccactgcaacaactggcatatctggcgattatgtacgcgctggtaccgctgttaatc<br />
atcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgc<br />
tgcatatggcgcttgctatcatcggcttactgtttatttgcgcgcatctctatctgtgtac<br />
tcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgtcatcgt<br />
accgcgccgcgcggggataagtccgccgtctga <br/><br />
<br />
<br />
<!------------- sequence data-------------><br />
</div><br />
<br/><br />
<br />
<br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 6/16--Checked on spotted cell survival assay, collected MOPS minimal media materials & started making component solutions<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b> Minimal Medium Work </b> <br/><br />
In Jay Keasling's work with thiosulfate reductase he used a variation of MOPS minimal medium designed to avoid background metal presence from interfering with measurement of bacterial metal removal potential. Began making the various necessary component solutions as detailed in this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/><br />
<b>Survival of bacteria in copper growth assay </b> <br/><br />
Previously had plated a drop of each culture from the narrow concentration range copper growth assay on 6/14 to see if the cultures that had stopped growing/never grew were still alive. Observed the following results, where g signifies growth and - signifies no growth:<br/><br />
<table><br />
<tr><br />
<td>cell line</td><br />
<td>0 M Cu<sup>2+</sup></td><br />
<td>500 uM Cu<sup>2+</sup></td><br />
<td>600 uM Cu<sup>2+</sup></td><br />
<td>700 uM Cu<sup>2+</sup></td><br />
<td>800 uM Cu<sup>2+</sup></td><br />
<td>900 uM Cu<sup>2+</sup></td><br />
<td>1 mM Cu<sup>2+</sup></td><br />
<td>1.5 mM Cu<sup>2+</sup></td><br />
<td>2 mM Cu<sup>2+</sup></td><br />
<td>3 mM Cu<sup>2+</sup></td><br />
<td>4 mM Cu<sup>2+</sup></td><br />
<td>1 M Cu<sup>2+</sup></td><br />
</tr><br />
<tr><br />
<td>DH5alpha</td><br />
<td>- </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>- </td><br />
<td>g </td><br />
<td>- </td><br />
<td>- </td><br />
<td>g </td><br />
<td>g </td><br />
<td>- </td><br />
</tr><br />
<tr><br />
<td>LE392</td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>- </td><br />
</tr><br />
</table><br />
It is unsurprising that all cells should die at 1 M copper sulfate concentrations, but the lack of growth of DH5alpha at 0 M, 900 uM, etc. seems anomalous, given that the cells clearly survived at higher copper levels and may reflect an insufficient amount of culture spotted in those cases. <br/><br />
<i>This information is also recorded on page 11 of the hard copy lab notebook. </i> <br/><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 6/17--more minimal media work and meeting, started BL21 culture<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<b>Modified MOPS minimal medium</b><br/><br />
Finished creation of a stock solution according the this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/><br />
<b>Additional <i> E. coli </i> strain </b> <br/><br />
Began a culture of BL21 as another hardy option for future assays. <br/><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 6/18--Arrival of plasmid pSB74, transformation of Biobricks, & copper growth assays for BL21 strain. <br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<ul><br />
<li>Plasmid pSB74, which contains the thiosulfate reductase gene, arrived from Addgene, already in some unspecified <i>E coli</i>, so plated them out on an ampicillin LB plate and put in incubator at 37.</li> <br />
<li>Transformed constitutive promote J23114, IPTG-inducible promoter R0011, terminator B0015, and repressor C0012(for inducible promoter) into BL21 according to standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation protocol </a> and plated onto ampicillin LB plates</li><br />
<li>Also ran BL21 copper growth assays for wide and narrow concentrations ranges according to the modified protocol used on 6/14, starting with dilution of a culture of OD 0.873.</li><br />
</ul><br />
<i> The activities of this day are also recorded on pages 13-15 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<li><br />
<br />
Saturday 6/19--Redo of Biobrick transformations & analysis of BL21 copper growth assay<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Saturday -------------><br />
<b>Transformation troubles</b> <br/><br />
While the pSB74 culture grew, none of the BL21 transformants from 6/19 did, so repeated the transformations of Biobricks B0015, R0011, C0012,and J23114 into LE392 cells, once again following this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation protocol</a> and plating onto ampicillin LB plates. The remaining transformant solutions were set aside in case of another failure.<br/><br />
<b>BL21 copper growth assay analysis</b> <br/><br />
Retrieved the following data regarding BL21's growth in copper solution: <br/><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f6/Yale-bl21.jpg" /><br />
</div><br />
It appears that BL21 is slightly more sensitive to copper than the other two strains, since 3 mM levels of copper(II) sulfate are enough to almost completely inhibit BL21's growth. <br/><br />
<i> The activities of this day are also recorded on page 15 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Sunday 6/20--Observed growth of all transformants from 6/19, so used them and the culture containing pSB74 to inoculate 5 mL liquid cultures in LB with ampicillin. Left to grow overnight on shaker at 37˚C for miniprep the following morning. <br/><br />
<i> The activities of this day are also recorded on page 16 of the hard copy lab notebook </i><br />
<br />
</li><br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
</ul><br />
</p><br />
</div><br />
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</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/NotebookTeam:Yale/Our Project/Notebook2010-10-28T02:47:55Z<p>Litagaki: </p>
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<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
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<div id="right-col"><br />
<h5><br />
lab notebook: week 1 (6/7 - 6/13)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 6/7--We successfully defend our project to our sponsors(yay!)<br />
<br />
</li><br />
<br />
<li><br />
<br />
Wednesday 6/9 --First day in lab!--got set up & started cultures of two <i> E. coli </i> strains<br />
<br />
</li><br />
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<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday-------------><br />
Began growing two strains of E. coli--LE392 chosen as a generally healthy option, and DH5alpha chosen because Keasling et. al. used it in their thiosulfate reductase work.<sup>1</sup> <br/><br />
<ul><br />
<br />
<li>Spread samples of LE392 (Grindley lab) and DH5alpha (Modis lab) on LB plates and left to incubate overnight at 37 </li><br />
<ul><br />
<sup>1</sup> Bang S. W., Clark D. S., Keasling J. D. Cadmium, lead, and zinc removal by expression of thiosulfate reductase gene from Salmonella typhimurium in Escherichia coli. Biotechnol. Lett. 2000; 22: 1331–1335,<br />
<!------------- Wednesday -------------><br />
</div><br />
<li><br />
<br />
Thursday 6/10--inoculated liquid cultures in AM, made up different copper sulfate & LB solutions, then ran an assay measuring bacterial growth in the presence of copper, trying both strains at three different initial ODs and measuring for four hours <br />
</li><br />
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<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
<h4> Baseline Wide Concentration Range Copper Growth Assay </h4><br />
<br />
Set out to find baseline growth exhibited by the E. coli strains being used when grown in a variety of copper concentrations. This will serve as a basis for comparison once copper tolerance has been engineered. Chose a range from the very, very low (sure to have no effect) to the very, very high (surely lethal).<br />
Set-up<br />
<ul><br />
<br />
<li>Studies were conducted in a 96-well plate, and growth was measured spectrophotometrically with a plate reader.</li><br />
<li>12 concentrations of copper(II) sulfate/LB solution were used with final (post-E. coli addition) copper(II) sulfate concentrations of 1 M, 0.1 M, 50 mM, 10 mM, 1 mM, 500 uM, 250 uM, 100 uM, 10 uM, 1 uM, 0.1 uM, and 0 uM, and each concentration was given its own column in the well plate. A row of cells with just the copper solutions was included to function as blanks </li><br />
</ul><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/3/30/Yale-well-plate.jpg" /><br />
<div id="caption">layout of 96-well plate</div><br />
</div><br />
<ul><br />
<li> Each of the two E. coli strains (LE392 and DH5alpha) was grown with each of the copper concentrations from three different starting densities. These three initial densities (OD 0.075,OD 0.0375, and OD 0.01875) were chosen so that mid-log phase would be reached at 1.5 hr, 2 hr. and 2.5 hr respectively after the start of the trial.</li><br />
<li> Absorption measurements were taken approximately every 10 minutes for four hours. Lack of a 600 nm filter necessitated measurement at 540 nm, a wavelength at which copper (II) sulfate absorbs, but the use of the appropriate blanks prevented this from interfering with data collection. </li><br />
<li>Samples were kept at 37 with alternated minutes of orbital shaking and stillness (plate reader does not allow continual shaking).</li><br />
</ul><br />
<br />
<h4> Results & Conclusions </h4><br />
<br /><br />
<div align="center" style="margin: -5px" ><img src="https://static.igem.org/mediawiki/2010/3/37/Yale-dh5alpha.png" /></div> <br />
<br /><br />
<ul><br />
<li> Both cells lines exhibit very similar copper tolerances, growing without noticeable effect at concentrations up to 500 uM, experiencing slowed growth at 1 mM, and no growth at 10 mM. </li><br />
<li> More trials are necessary at concentration ranges between 500 uM and 10 mM to determine more exactly the maximum copper tolerance of these cell lines. </li><br />
<li> After four hours of growth the healthier cells were still growing very rapidly. Future assays will extend recording time to five hours in an effort to capture the plateau region </li><br />
</ul><br />
<br />
<i> This work is also documented on pages 1-5 in the hard copy of the lab notebook </i><br />
<!------------- Thursday-------------><br />
</div><br />
<br />
<li><br />
<br />
Friday 6/11--Ran another growth assay with middling concentrations based on results of first, but bacteria failed to grow well--perhaps because allowed to overgrow prior to assay?<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4> Further Copper Growth Assays—Narrow Concentration Range </h4><br />
Plan to do more assays of bacterial growth in the presence of copper to determine the exact threshold at which growth is affected. Will repeat yesterday’s assay with a more targeted concentration range and a longer growth time. <br />
<ul><br />
<li>Once again inoculated liquid cultures of DH5alpha and LE392 in 5 mL LB using bacteria from plates established on 6/9 and put on shaker to grow at 37˚C </li><br />
<li>Let grow for approximately 6 hours, by which point the DH5alpha have reached an OD (measured at 600 nm) of 0.915and the LE392 have reached OD 1.420. </li><br />
<li>Diluted them to the three initial ODs chosen on 6/10 in a variety of copper (II) sulfate concentrations chosen to give more detailed data than the wide concentration range trial. The concentrations chosen were 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, 1.25 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM </li><br />
<li>Using these concentrations, ran the copper growth assay overnight using the same protocol as the previous day (link), except that the plate reader took readings for five hours instead of four to better capture all of the growth phase behavior.</li><br />
</ul><br />
<i> These notes can also be found on pages 6-8 of the hard copy lab notebook </i><br />
<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
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</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T02:47:11Z<p>Litagaki: </p>
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<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T02:46:28Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:45:09Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">H<sub>2</sub>S production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">Cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">Cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. According to their results, thiosulfate reductase encoded in the plasmid pSB74 showed the highest activity catalytic activity, so we obtained phsABC from the plasmid pSB74. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, inserted phsABC into B0015:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the phsABC gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> phsABC gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SRB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T02:43:13Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T02:41:35Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<h4>Ligation of Q04121 and phsABC/B0015 construct </h4><br />
<ul><br />
<li>Given that the DNA concentration of the 5.8 kb Q04121 insert is 20 ng/uL and that of the 7.8 kb vector is 19.3 ng/uL, calculate that 2.15 uL of insert is stoichiometric equivalent of 3 uL of vector. Based on this set-up the following ligation reactions and let them run for 8 hours at 16˚C before a 20 minute heat-kill. </li><br />
<b> Control ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, and 14 uL of water. <br/><br />
<b> 2:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 4.3 uL insert, and 9.7 uL of water. <br/><br />
<b> 5:1 Insert:vector ligation </b> 2 uL of ligase buffer, 1 uL T4 ligase, 3 uL of vector, 10.8 uL of insert, and 3.2 uL of water. <br/><br />
<li> After the ligations were heat-killed, transformed them into Top10 supercompetent cells using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> and a pUC19 control. Plated on kanamycin plates and left in incubator overnight. </li><br />
</ul><br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--No colonies visible from 7/29 transformation, so returned plates to incubator.<br />
<br />
</li><br />
<br />
<br />
<li><br />
<br />
Saturday 7/31--Plates from ligation transformation showed growth.<br />
<br />
</li><br />
<a id="link" href="#" onclick="toggle_visibility('saturday'); return false;">See more</a><br />
<div style="display:none" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4>Second ligation transformants </h4><br />
<ul><br />
<li>The pUC19 and control ligation plates both showed many colonies, but the 2:1 ligation showed even more, while the 5:1 ligation showed 18 large colonies. </li><br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:24:16Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. According to their results, thiosulfate reductase encoded in the plasmid pSB74 showed the highest activity catalytic activity, so we obtained phsABC from the plasmid pSB74. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, inserted phsABC into B0015:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the phsABC gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> phsABC gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SRB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:23:43Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. According to their results, thiosulfate reductase encoded in the plasmid pSB74 showed the highest activity catalytic activity, so we obtained phsABC from the plasmid pSB74. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, inserted phsABC into B0015:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the phsABC gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> phsABC gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SFB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:22:02Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. According to their results, thiosulfate reductase encoded in the plasmid pSB74 showed the highest activity catalytic activity, so we obtained phsABC from the plasmid pSB74. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, inserted phsABC into B0015:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SFB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:21:02Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. According to their results, thiosulfate reductase encoded in the plasmid pSB74 showed the highest activity catalytic activity, so we obtained phsABC from the plasmid pSB74. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, phsABC to B0015:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SFB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:18:16Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained from Dr. Jay Keasling laboratory at University of California, Berkeley. According to their results, Thiosulfate Reductase encoded in the plasmid pSB74 showed the highest activity, so we obtained phsABC from the plasmid pSB74. E. coli DH5α strain were used for plasmid manipulation.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, phsABC to B0015:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SFB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:16:20Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained from Dr. Jay Keasling laboratory at University of California, Berkeley. According to their results, Thiosulfate Reductase encoded in the plasmid pSB74 showed the highest activity, so we obtained phsABC from the plasmid pSB74. E. coli DH5α strain were used for plasmid manipulation.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b>Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(3) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, we added the promoter to phsABC:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SFB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/MethodsTeam:Yale/Our Project/Methods2010-10-28T02:13:14Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></b></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">plasmid</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/h2s production ">h2s production</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu growth assay">cu growth assay</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/cu localization">cu localization</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Methods/parts">parts</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
experimental methods<br />
</h5><br />
<!------------- METHODS: NEEDS TO BE EDITED-------------><br />
<p><br />
Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase. <br />
</p><br />
<b><br />
(1) phsABC gene and vector<br />
</b><br />
<p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/f/f8/Phsplasmiddiagram.png/800px-Phsplasmiddiagram.png" " width="568" height="70" /> <br />
<div id="caption">phsABC in pSB74</div><br />
</div><br />
</p><br />
<p><br />
This central component encodes Thiosulfate Reductase. The gene phsABC was obtained from Dr. Jay Keasling laboratory at University of California, Berkeley. According to their results, Thiosulfate Reductase encoded in the plasmid pSB74 showed the highest activity, so we obtained phsABC from the plasmid pSB74. E. coli DH5α strain were used for plasmid manipulation.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/0/09/Yale-keasling1.png" /><br />
<div id="caption">Table from Keasling’s research: comparison of Thiosulfate reductase activity</div><br />
</div><br />
<br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ad/Yale-keasling2.png" /><br />
<div id="caption">Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.</div><br />
</div><br />
<br />
<b><br />
(2) Biobrick Promoter</b> <p>Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .</p><br />
<br /><br />
<b><br />
(3) Promoter B0034</b><br />
<br /> <br /><br />
<img src="https://static.igem.org/mediawiki/2010/a/a6/Yale-promoter.png" /><br />
<p><br />
Biobrick Part:BBa_Q04121.<br />
<br />Length 1370 bp<br />
<br />IPTG-induced (regulatory)</p><br />
<br /><br />
<b><br />
(4) Biobrick Terminator</b> The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.<br />
<br /><br />
<br /><br />
<b><br />
Restriction Enzyme Sites:</b><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ed/Yale-restriction-enzyme.png" /><br />
</div><br />
<div id="caption">The sites are shown in <a id="link" href="http://www.neb.com/">New England BioLabs Inc.</a></div><br />
<br />
<b>Plasmid Construction</b><br />
<p>The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene. </p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/6/60/Yale-plasmid-construction.png" /><br />
</div><br />
<br />
<p>By using this ligation method, we added the promoter to phsABC:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /></div><br />
<br /><br />
<br /><br />
<br />
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a><br />
<div style="display:none;" id="background"><br />
<p><br />
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br><br />
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br><br />
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br><br />
• 1995, phs locus of Salmonella chromosome located, sequenced, and confered H2S production to E.Coli. </br></br><br />
– Sequence similar to reductases in other bacteria</br></br><br />
– Later confirmed to contain structural gene for thiosulfate reductase</br></br><br />
• Since before 1970 people have observed bacteria producing H2S. The earliest samples were taken from patients (Stoleru and Bouanehaud of Institut Pasteur, 1972 and 1975). Better yet, they showed that H2S production was mediated by a plasmid! A later study in 1978 by Jones et al of Texas Tech characterized another one of these plasmids, saw that it could be used in and transferred between E. Coli as well! </br></br><br />
• In 1987 an extensive review was written collecting lots of existing research about sulfate-reducing bacteria (SFB). In this broad review of SFB, it mentioned that sulfate reductases are membrane bound. Possibly useful for us, purified samples of thiosulfate reductase can also apparently be controlled, stopped in the presence of NADH , NADPH, and cysteine. Anyhow, the main development in this paper showed that thiosulfate-reduction ability is fairly common, usually used by anaerobic bacteria as an energy source (although some aerobic variants have been reported) and that one of the key players is thiosulfate reductase, which by this point had been isolated and had some characterization available. If required, there are lots of other sulfur-reducing pathways that are common as well, with a notable one being the reduction of tetrathionate.</br></br><br />
• Perhaps not surprising, the same authors of the review paper actually in the same year published a paper on the phs gene mediating hydrogen-sulfide production! Better yet, this gene is not coupled to methyl-viologen, which is common in anaerobic bacteria! score! Of note, the authors write that the phs gene is not actually the structural gene itself, but instead codes for a regulatory protein important for the reduction of thiosulfate to H2S. This paper also looked at different ways to optimize H2S production. </br></br><br />
• Something happened over a decade (particularly, see 1995 paper by Erika Barrett analyzing the phs sequence), because by 1999 a study from UC Berkeley, in Keasling lab, had the actual structural genes! In a couple of studies (1999-2000) they optimized the phs H2S production system to produce lots of H2S and remove heavy metals from solution! Optimization of their system is given in the slide to the left </br></br><br />
• Keasling lab, used phs gene sequences transformed into DH5α for bioremediation studies (2000). </br></br><br />
• <br />
• Optimized reading frame and plasmid for H2S production </br></br><br />
• Optimized conditions for precipitation of metals<br />
• (zinc, lead, and cadmium) </br></br><br />
</div><br />
<br />
<!------------- METHODS: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T01:49:57Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest</li><br />
<li>Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water. </li><br />
<li>phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.</li><br />
<li> Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--First attempt at Q04121 ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday short content<br />
<br />
</li><br />
<a id="link" href="#" onclick="toggle_visibility('saturday'); return false;">See more</a><br />
<div style="display:none" id="saturday"><br />
<!------------- Saturday -------------><br />
Extra content<br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday short content<br />
<br />
</li><br />
<a id="link" href="#" onclick="toggle_visibility('sunday'); return false;">See more</a><br />
<div style="display:none" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-28T01:34:11Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4>Miniprep & sequencing samples of likely ligation sucesses </h4><br />
<ul><br />
<li>Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">vacuum manifold protocol</a> and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.</li><br />
<li> Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction. </li><br />
</ul><br />
<i> This day's work is also recorded on pages 81-82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--Diagnostic double digest of likely ligations<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<br />
<!------------- Tuesday -------------><br />
<h4>Diagnostic Double Digest of Supposed Ligations </h4><br />
<ul><br />
<li>While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success. </li><br />
<li> Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless. </li><br />
</ul><br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28--Sequencing confirms ligation success!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<!------------- Wednesday -------------><br />
<h4>Sequencing success at last! </h4><br />
<ul><br />
<li> Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015. </li><br />
<a id="link" href="javascript:ReverseDisplay('sequence data')">See/hide sequence data</a><br />
<div style="display:none;" id="sequence data"><br />
<br />
<!------------- atgc -------------><br />
>sequence of desired phsABC/B0015 construct (in <br />
<br />
<!------------- atgc -------------><br />
</ul><br />
<h4>Beginning of second ligation efforts </h4><br />
<ul> <br />
<li>With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with sequential double digests of Q04121 and the phsABC/B0015 construct</li><br />
</ul><br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday short content<br />
<br />
</li><br />
<a id="link" href="#" onclick="toggle_visibility('saturday'); return false;">See more</a><br />
<div style="display:none" id="saturday"><br />
<!------------- Saturday -------------><br />
Extra content<br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday short content<br />
<br />
</li><br />
<a id="link" href="#" onclick="toggle_visibility('sunday'); return false;">See more</a><br />
<div style="display:none" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
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</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-28T00:50:44Z<p>Litagaki: </p>
<hr />
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{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract"> gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" /><br />
</div><br />
<br /><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a9/Yale-self-ligation.jpg" /><br />
</div><br />
<br /><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
</ul><br />
<h4>Ligation using serial digestion products </h4><br />
<ul><br />
<li>Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the <a href=" https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> standard protocol </a>, then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015. </li><br />
<li> Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA </li><br />
<b>T4 ligase control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water <br/><br />
<b>T4 ligase 2:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water <br/><br />
<b>T4 ligase 6:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC <br/><br />
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/><br />
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/><br />
<br />
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li><br />
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/><br />
<b>2:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water <br/><br />
<b>2:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water <br/><br />
<b>6:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC<br/><br />
<b>6:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC <br/><br />
Molar ratios are approximate.<br/><br />
<li> All ligations were allowed to run overnight at 16˚C. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--Ligation Transformations<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<b> Q02141 transformation </b> <br/><br />
<ul><br />
<li><br />
No growth was observed on the Q04121 plates--may need to find better competent cells. </li><br />
</ul><br />
<h4> Transformation of Varied Ligation Efforts </h4><br />
<ul><br />
<li> Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C. </li><br />
<li> After previous ligation troubles, have invested in commercial grade ultracompetent cells. Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a>, also transforming the pUC19 standard that came with the cells. All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--Results of ligation transformations<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Ligation Transformants </h4><br />
<ul><br />
<li> The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable. The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all. In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate. </li><br />
<li> Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and mixing a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the resulting PCR reaction on the "VR" protocol of the thermocycler. </li><br />
<li> Also started liquid cultures of each of the colonies in ampicillin LB. </li><br />
<li> The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24 </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4> Gel of Colony PCR </h4><br />
<ul><br />
<li> Ran the twenty-four colony PCR reactions from 7/23 on a single massive 1.0% agarose gel vs a 1 kb ladder at 90 V.</li><br />
Ladder is very faint in the leftmost lane and the twenty-four reactions are in order from left to right. Despite the faintness, one can see that samples 5, 12, 14, 15, 20, 21 and 24 appear to have the desired 3.6 kb fragment indication a successful ligation.</li><br />
</ul><br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--Inoculate cultures of likely ligations successes for miniprep on 7/26<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
<b> Inoculation of liquid cultures </b> <br/><br />
<ul><br />
<li> Inoculated liquid ampicillin in LB cultures from the colonies shown to contain successful ligations by the 9/25 gel of colony PCR. Also inoculated a culture of Q04121 for overnight growth, adding kanamycin to a concentration of 50 ng/uL. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
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</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-28T00:43:48Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract"> gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" /><br />
</div><br />
<br /><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a9/Yale-self-ligation.jpg" /><br />
</div><br />
<br /><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
</ul><br />
<h4>Ligation using serial digestion products </h4><br />
<ul><br />
<li>Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the <a href=" https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> standard protocol </a>, then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015. </li><br />
<li> Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA </li><br />
<b>T4 ligase control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water <br/><br />
<b>T4 ligase 2:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water <br/><br />
<b>T4 ligase 6:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC <br/><br />
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/><br />
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/><br />
<br />
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li><br />
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/><br />
<b>2:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water <br/><br />
<b>2:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water <br/><br />
<b>6:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC<br/><br />
<b>6:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC <br/><br />
Molar ratios are approximate.<br/><br />
<li> All ligations were allowed to run overnight at 16˚C. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--Ligation Transformations<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<b> Q02141 transformation </b> <br/><br />
<ul><br />
<li><br />
No growth was observed on the Q04121 plates--may need to find better competent cells. </li><br />
</ul><br />
<h4> Transformation of Varied Ligation Efforts </h4><br />
<ul><br />
<li> Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C. </li><br />
<li> After previous ligation troubles, have invested in commercial grade ultracompetent cells. Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a>, also transforming the pUC19 standard that came with the cells. All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--Results of ligation transformations<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Ligation Transformants </h4><br />
<ul><br />
<li> The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable. The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all. In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate. </li><br />
<li> Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and mixing a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the resulting PCR reaction on the "VR" protocol of the thermocycler. </li><br />
<li> Also started liquid cultures of each of the colonies in ampicillin LB. </li><br />
<li> The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24 </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4> Gel of Colony PCR </h4><br />
<ul><br />
<li> Ran the twenty-four colony PCR reactions from 7/23 on a single massive 1.0% agarose gel vs a 1 kb ladder at 90 V.</li><br />
Ladder is very faint in the leftmost lane and the twenty-four reactions are in order from left to right. Despite the faintness, one can see that samples 5, 12, 14, 15, 20, 21 and 24 appear to have the desired 3.6 kb fragment indication a successful ligation.</li><br />
</ul><br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--Inoculate cultures of likely ligations successes for miniprep on 7/26<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
<b> Inoculation of liquid cultures </b> <br/><br />
<ul><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-28T00:38:51Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
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<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract"> gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" /><br />
</div><br />
<br /><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a9/Yale-self-ligation.jpg" /><br />
</div><br />
<br /><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
</ul><br />
<h4>Ligation using serial digestion products </h4><br />
<ul><br />
<li>Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the <a href=" https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> standard protocol </a>, then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015. </li><br />
<li> Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA </li><br />
<b>T4 ligase control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water <br/><br />
<b>T4 ligase 2:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water <br/><br />
<b>T4 ligase 6:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC <br/><br />
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/><br />
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/><br />
<br />
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li><br />
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/><br />
<b>2:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water <br/><br />
<b>2:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water <br/><br />
<b>6:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC<br/><br />
<b>6:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC <br/><br />
Molar ratios are approximate.<br/><br />
<li> All ligations were allowed to run overnight at 16˚C. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--Ligation Transformations<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<b> Q02141 transformation </b> <br/><br />
<ul><br />
<li><br />
No growth was observed on the Q04121 plates--may need to find better competent cells. </li><br />
</ul><br />
<h4> Transformation of Varied Ligation Efforts </h4><br />
<ul><br />
<li> Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C. </li><br />
<li> After previous ligation troubles, have invested in commercial grade ultracompetent cells. Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a>, also transforming the pUC19 standard that came with the cells. All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--Results of ligation transformations<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Ligation Transformants </h4><br />
<ul><br />
<li> The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable. The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all. In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate. </li><br />
<li> Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and mixing a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the resulting PCR reaction on the "VR" protocol of the thermocycler. </li><br />
<li> Also started liquid cultures of each of the colonies in ampicillin LB. </li><br />
<li> The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24 </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
<h4> Gel of Colony PCR </h4><br />
<ul><br />
<li> Ran the twenty-four colony PCR reactions from 7/23 on a single massive 1.0% agarose gel vs a 1 kb ladder at 90 V.</li><br />
Ladder is very faint in the leftmost lane and the twenty-four reactions are in order from left to right. Despite the faintness, one can see that samples 5, 12, 14, 15, 20, 21 and 24 appear to have the desired 3.6 kb fragment indication a successful ligation. </li><br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-28T00:24:49Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract"> gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" /><br />
</div><br />
<br /><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a9/Yale-self-ligation.jpg" /><br />
</div><br />
<br /><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
</ul><br />
<h4>Ligation using serial digestion products </h4><br />
<ul><br />
<li>Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the <a href=" https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> standard protocol </a>, then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015. </li><br />
<li> Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA </li><br />
<b>T4 ligase control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water <br/><br />
<b>T4 ligase 2:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water <br/><br />
<b>T4 ligase 6:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC <br/><br />
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/><br />
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/><br />
<br />
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li><br />
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/><br />
<b>2:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water <br/><br />
<b>2:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water <br/><br />
<b>6:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC<br/><br />
<b>6:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC <br/><br />
Molar ratios are approximate.<br/><br />
<li> All ligations were allowed to run overnight at 16˚C. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--Ligation Transformations<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
<b> Q02141 transformation </b> <br/><br />
<ul><br />
<li><br />
No growth was observed on the Q04121 plates--may need to find better competent cells. </li><br />
</ul><br />
<h4> Transformation of Varied Ligation Efforts </h4><br />
<ul><br />
<li> Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C. </li><br />
<li> After previous ligation troubles, have invested in commercial grade ultracompetent cells. Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> transformation protocol </a>, also transforming the pUC19 standard that came with the cells. All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin. </li><br />
</ul><br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--Results of ligation transformations<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Ligation Transformants </h4><br />
<ul><br />
<li> The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable. The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all. In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate. </li><br />
<li> Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and mixing a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the resulting PCR reaction on the "VR" protocol of the thermocycler. </li><br />
<li> Also started liquid cultures of each of the colonies in ampicillin LB. </li><br />
<li> The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24 </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a><br />
<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-28T00:03:35Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract"> gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Yale-dual-purpose-gel.jpg" /><br />
</div><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
</ul><br />
<h4>Ligation using serial digestion products </h4><br />
<ul><br />
<li>Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the <a href=" https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> standard protocol </a>, then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015. </li><br />
<li> Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA </li><br />
<b>T4 ligase control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water <br/><br />
<b>T4 ligase 2:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water <br/><br />
<b>T4 ligase 6:1 insert:vector </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC <br/><br />
<b>Quick ligase control ligation</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water <br/><br />
<b>Quick ligase 2:1 insert:vector</b>--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC <br/><br />
<i>This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook </i><br />
<li> Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase. </li><br />
<b>control ligation </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water <br/><br />
<b>2:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water <br/><br />
<b>2:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water <br/><br />
<b>6:1 gel extraction product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC<br/><br />
<b>6:1 PCR cleanup product </b>--2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC <br/><br />
Molar ratios are approximate.<br/><br />
<li> All ligations were allowed to run overnight at 16˚C. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--<br />
<br />
</li><br />
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<br />
<!------------- Thursday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--<br />
<br />
<br />
</li><br />
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<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--<br />
<br />
</li><br />
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<!------------- Saturday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
</li><br />
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<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-27T23:44:05Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract " gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b>Q04121</b> <br/><br />
<ul><br />
<li> Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">standard transformation procedure </a> and plate on Kanamycin plates. </li><br />
</ul><br />
<h4>Gel of phsABC self-ligation efforts </h4><br />
<ul><br />
<li>Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.</li><br />
From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC <br/><br />
<br />
<li>The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end. </li><br />
<h4>Ligation using serial digestion products </h4><br />
<i>This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--<br />
<br />
</li><br />
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<br />
<!------------- Thursday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--<br />
<br />
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</li><br />
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<!------------- Friday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--<br />
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<!------------- Saturday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
</li><br />
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<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-27T23:24:11Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
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</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract " gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components<br />
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</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4>Self-ligation of singly digested phsABC fragments</h4><br />
<ul><br />
<li> Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results. </li><br />
</ul><br />
<h4>Serial digestion of ligation components </h4><br />
<ul><br />
<li> Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C. </li><br />
<li> Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before. </li><br />
</ul><br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--<br />
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</li><br />
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<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
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<li><br />
<br />
Thursday 7/22--<br />
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</li><br />
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<div style="display:none;" id="thursday"><br />
<br />
<!------------- Thursday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--<br />
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</li><br />
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<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--<br />
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</li><br />
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<div style="display:none;" id="saturday"><br />
<!------------- Saturday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
</li><br />
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<div style="display:none;" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
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</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_2Team:Yale/Our Project/Notebook/Week 22010-10-27T23:06:49Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 2 (6/14-6/20)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 6/14--Redo of <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook">6/11</a> assay of bacterial growth within a narrow copper(II) concentrations after analysis of data showed uniformly poor growth.<br />
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</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Work Continues </h4><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8f/Yale-growthfail.jpg" /><br />
</div><br />
<ul><br />
<li>As the growth assay of <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook">6/11</a> showed uniformly poor growth even at the lowest copper concentrations (see Dh5alpha growth above), redid it with care not to let the liquid cultures overgrow, a possible source of the cultures' previous poor performance. (add plot)</li><br />
<li>In redoing plate assay, changed copper concentration line-up slightly, eliminating 1.25 mM and 2.5 mM trials to allow room for the control concentrations, 0 and 1 M, and only worked with high because there was no real distinction in the three different OD trials done on 6/10. For simplicity, future assays will only use one starting OD (0.075) per strain. </li><br />
<li>During the repeat, noticed that DH5alpha liquid culture grown up for plate reader assay grew at a rate much slower than that of LE392, creating a need to keep diluting the LE392 solution with more LB so it would not overgrow before the DH5alpha was ready. In the end, the culture of LE392 used in the assay was diluted from an OD of 0.761 and the DH5alpha culture from an OD of 0.841.</li><br />
</ul><br />
<i> This day's work is also recorded on pages 8-10 of the lab notebook hard copy. </i><br />
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<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 6/15--Results and analysis of the 6/14 narrow concentration range growth assay as well as planning for the creation of a standard iGEM plasmid bearing the thiosulfate reductase operon (phsABC).<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> Results and Conclusions of Narrow Concentration Range Growth Assay of 6/14</h4><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/d/d1/Yale-dh5alpha-narrow.jpg" /><br />
</div><br />
<ul><br />
<li> Negative readings for the 4 mM samples suggest that there was some sort of irregularity with the blank solution, so will disregard those results. </li><br />
<li> Can see growth really starts to slow down at 3 mM concentrations of Cu<sup>2+</sup> </li><br />
<li>It’s unclear whether the cells at the higher copper concentrations die or simply stop growing. Spot a drop from each well of the plate onto a LB agar plate and incubate overnight at 37˚C. Will check for growth to determine survival of different strains at different copper concentrations.</li><br />
</ul><br />
<i>This information is also recorded on page 11 of the hard copy lab notebook. </i> <br/><br />
<h4> Plasmid Ligation Strategy</h4><br />
<b> The Plan for phs plasmid creation </b> <br/><br />
The phs operon has three genes of interest to us, A, B, and C, that code for different protein products and together are responsible for hydrogen sulfide production. We will use these genes to create a number of different plasmids described below. <br/><br />
<b> Plasmids to be made: </b> <br/><br />
Plasmid 1 gives our basic thiosulfate reductase pathway under IPTG control. Plasmid 5 is a light-controlled analog that we will make if we can find a suitable light-inducible promoter. Plasmid 2 is a promoter-less “generator” to be archived so other teams can use it. Plasmids 3 & 4 are a set and together give the entire pathway. The idea is that since protein production takes a while we will make only part of the pathway inducible. The cell will constitutively produce the A & B products, so after the light hits only the small C protein remains to be made. <br/><br />
<table border="1"><br />
<tr><br />
<td>Product</td><br />
<td>Promoter</td><br />
<td>Gene</td><br />
<td>Terminator</td><br />
<td>Plasmid Vector</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>IPTG-inducible BBa_R0011(well 6G plate 1)</td><br />
<td> phsABC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>none)</td><br />
<td> phsABC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>Constitutive BBa_J23114 (well 20I plate 1)</td><br />
<td> phsAB </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1T3 (Tet resistance)</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>Light-inducible</td><br />
<td> phsC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>light-inducible</td><br />
<td> phsABC </td><br />
<td> BBa_B0015 </td><br />
<td> pSB1C3 (chloramphenicol resistance)</td><br />
</tr><br />
<tr><br />
</table> <br />
*This plan was eventually altered, as promoter choices were altered and no light-inducible promoter was found. <br/><br />
<br /><br />
<div id="right"><br />
<img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" /><br />
</div><br />
To make these plasmids, we will rely on the standard iGEM assembly protocol involving restriction enzymes EcoRI, XbaI, PstI, and SpeI shown in the diagram at right, but in the first ligation the B0015 terminator will take the place of C0010 and the phsABC gene in pSB74 will take the place of B0034. <br/><br />
<br />
<b> Steps to make these plasmids </b> <br/><br />
1. Initial Amplification—transform and amplify terminator, promoter, and gene DNA <br/><br />
2. Gene-Terminator linkage—Digest terminator with EcoRI and XbaI, digest PCR product gene with EcoRI and SpeI, and ligate together to get gene and terminator in nonstandard plasmid <br/><br />
3. Creation of Standard Generator—digest step 2 product with EcoRI and PstI and ligate into standard iGEM plasmid pSB1C3 to get the generator <br/><br />
4. Addition of promoter—Digest promoter with EcoRI and Spe--digest generator vector with EcoRI and XbaI, and ligate together<br/><br />
<br />
<h4> Primer design for PCR amplification of the phsABC gene from background vector pSB74 </h4><br />
Given the size of phsABC, introducing it in pieces with AB on one plasmid and C on a second might enhance expression. For this purpose, we have designed forward primers that include the EcoRI and XbaI cut sites and an RBS to go at the beginning of A and C as well as reverse primers with a SpeI site to go at the end of B and C. <br/><br />
The following primers were designed based on the phsABC sequence information (see below) after having confirmed that the BioBrick restriction enzyme cut sites are not present in the phsABC sequence. <br/><br />
<br />
N-terminal primer for phsABC: 5’ GCA GAATTC GCGGCCGC T TCTAGA G AGGAGGTTTAT ATG AGC ATT AGT CGT CG <br/><br />
C-terminal primer for phsAB: 5’ AGC ACTAGT A TCA TGC TTT ACC CTC CTG ATG GAC <br/><br />
N-terminal primer for phsC: 5’ GCA GAATTC GCGGCCGC T TCTAGA G AGGAGGTTTAT ATG AAT ACT ATC TGG GG <br/><br />
C-terminal primer for phsABC: 5’ AGC ACTAGT A TCA GAC GGC GGA CTT ATC CCC <br/><br />
<br />
<a id="link" href="javascript:ReverseDisplay('sequence')">See/hide phsABC sequence </a><br />
<div style="display:none;" id="sequence"><br />
<br />
<!------------- sequence data-------------><br />
phsA STM2065 <br/><br />
>sp|P37600|PHSA_SALTY Thiosulfate reductase OS=Salmonella typhimurium GN=phsA PE=3 SV=2 <br/><br />
MSISRRSFLQGVGIGCSACALGAFPPGALARNPIAGINGKTTLTPSLCEMCSFRCPIQAQ VVNNKTVFIQGNPSAPQQGTRICARGGSGVSLVNDPQRIVKPMKRTGPRGDGEWQVISWQ QAYQEIAAKMNAIKAQHGPESVAFSSKSGSLSSHLFHLATAFGSPNTFTHASTCPAGKAI AAKVMMGGDLAMDIANTRYLVSFGHNLYEGIEVADTHELMTAQEKGAKMVSFDPRLSIFS SKADEWHAIRPGGDLAVLLAMCHVMIDEQLYDASFVERYTSGFEQLAQAVKETTPEWAAA QADVPADVIVRVTRELAACAPHAIVSPGHRATFSQEEIDMRRMIFTLNVLLGNIEREGGL YQKKNASVYNKLAGEKVAPTLAKLNIKNMPKPTAQRIDLVAPQFKYIAAGGGVVQSIIDS ALTQKPYPIKAWIMSRHNPFQTVTCRSDLVKTVEQLDLVVSCDVYLSESAAYADYLLPEC TYLERDEEVSDMSGLHPAYALRQQVVEPIGEARPSWQIWKELGEQLGLGQYYPWQDMQTR QLYQLNGDHALAKELRQKGYLEWGVPLLLREPESVRQFTARYPGAIATDSDNTYGEQLRF KSPSGKIELYSATLEELLPGYGVPRVRDFALKKENELYFIQGKVAVHTNGATQYVPLLSE LMWDNAVWVHPQTAQEKGIKTGDEIWLENATGKEKGKALVTPGIRPDTLFVYMGFGAKAG<br />
AKTAATTHGIHCGNLLPHVTSPVSGTVVHTAGVTLSRA <br/><br/><br />
<br />
>phsA <br/><br />
atgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcg ctgggcgcttttccgcccggggcgctggcgcgcaacccgattgccggcattaacggtaaa accacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcag gtagtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacg cgcatatgcgccaggggcgggagcggcgtcagcctggtcaatgacccgcaacggattgta aaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaa caggcttaccaggaaatcgcggcgaaaatgaatgccatcaaagcgcagcatggccccgag agcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacg gcctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccatt gcggcaaaagtgatgatgggcggcgatctggcgatggatatcgctaacacgcgctatctg gtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatg accgcgcaggagaagggggccaaaatggtgagcttcgatccgcgtttgtcgatattttcc agcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcg atgtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttatacc agcggatttgaacagttagcacaggcggtaaaagagacgacgccggaatgggccgccgcg caggccgatgtcccagccgacgttattgtccgggtgacacgcgaactggctgcctgcgcg cctcacgctattgtcagtcctggtcatcgcgcgacgttctcgcaggaagagatcgatatg cggcgtatgatttttacgcttaatgtgctgctcggtaatattgagcgcgaaggcgggcta tatcagaaaaaaaacgcgtctgtttacaataaactggccggagaaaaggtcgcgccaacg ctggcgaaactcaacattaaaaatatgccgaaaccgacggcgcaacgcatcgatttggtc gcaccgcagtttaaatatatcgccgctggcggcggcgtggtgcaaagcattattgactcg gcgttaacccagaagccttacccgataaaggcgtggattatgtcgcggcataatcctttc cagaccgtcacctgtcgttcggacctggtaaaaaccgttgagcaactggatctggtggtc agctgcgatgtctatttgagcgagagcgcggcatatgccgactatctgctgccggaatgc acctatctcgaacgggacgaagaggtatccgatatgtcgggactgcacccggcttacgct ctgcgccaacaggtcgtagagccgattggcgaggcgcgtccgagttggcaaatctggaaa gaacttggcgagcagttgggattagggcagtactatccgtggcaggatatgcagacgcgc caactctatcagttgaacggcgaccatgccttagcgaaggaactgcgacagaaagggtat ctcgaatggggcgttccgctgctattacgcgaaccagaatccgttcgtcagtttacggcg cgttaccccggcgctatcgcgacggacagtgacaacacctatggcgaacagcttcgcttc aaatcgccctccggcaaaatcgaactttattccgcaaccctggaggaattgctccctggc tacggcgttccgcgcgttcgtgactttgcgctgaaaaaagagaatgagctttacttcatt cagggcaaggtggccgtgcataccaatggcgcgacgcagtacgtacctttactcagcgag ctaatgtgggataacgcggtctgggttcatccgcaaacggcgcaagaaaaaggcattaag accggcgatgagatctggctggaaaatgccacgggtaaagagaaaggtaaggcgctggtg acgcccggtatccgcccggacacgctttttgtctatatgggatttggcgctaaagctggg gccaaaacggcggcgacgacacacggtatccactgcggaaatttactgccgcacgtgacg agtccggtatccggtacggtagtgcataccgcaggcgtgacgctgagccgggcatga <br/><br/><br />
<br />
phsB STM2064 <br/><br />
>sp|P0A1I1|PHSB_SALTY Thiosulfate reductase electron transport protein phsB OS=Salmonella typhimurium GN=phsB PE=4 SV=1 <br/> MNHLTNQYVMLHDEKRCIGCQACTVACKVLNDVPEGFSRVQVQIRAPEQASNALTHFQFV RVSCQHCENAPCVSVCPTGASYRDENGIVQVDKSRCIGCDYCVAACPFHVRYLNPQTGVA DKCNFCADTRLAAGQSPACVSVCPTDALKFGRLDESEIQRWVGQKEVYRQQEARSGAVSL <br />
YRRKEVHQEGKA <br/><br/><br />
<br />
>phsB <br/><br />
Atgaatcatttaacgaatcagtacgtcatgctgcatgatgaaaaacgttgtatcggctgc<br />
caggcgtgtaccgttgcctgcaaagtgcttaatgacgtaccggagggatttagccgcgta<br />
caggtacaaattcgcgcccctgaacaggcatccaacgcactaacccattttcaatttgtc<br />
cgcgtctcctgtcagcactgtgaaaatgcgccatgtgtcagcgtttgtcctaccggagcg<br />
tcatatcgtgatgaaaacgggatcgtgcaggtggataaatcgcgctgtattggctgcgat<br />
tattgtgttgccgcgtgtcctttccatgtgcgctatttgaatccgcaaaccggtgtcgcc<br />
gacaagtgtaacttctgcgccgacacgcggttggcggctggccagtctccggcgtgcgta<br />
tccgtttgcccaacggacgcgctgaaattcggcagactggatgagagcgagatccagcgc<br />
tgggtcggtcagaaagaggtctatcgccagcaggaggcgcgtagcggcgcggtcagtctg<br />
taccgtcgtaaagaagtccatcaggagggtaaagcatga <br/><br/><br />
<br />
phsC STM2063 <br/><br />
>sp|P37602|PHSC_SALTY Thiosulfate reductase cytochrome B subunit OS=Salmonella typhimurium GN=phsC PE=4 SV=2 <br/><br />
MNTIWGAELHYAPDYWPLWLIYAGVVVLLMLVGLVIHALLRRMLAPKTAGGEEHRDYLYS LAIRRWHWGNALLFVLLLLSGLFGHFSLGPVALMVQVHTWCGFALLAFWVGFVLINLTTG NGRHYRVNFSGLVTRCIRQTRFYLFGIMKGEAHPFVATEQNKFNPLQQLAYLAIMYALVP LLIITGLLCLYPQVAGLGPVMLVLHMALAIIGLLFICAHLYLCTLGDTPGQIFRSMVDGY <br />
HRHRTAPRGDKSAV <br/><br/><br />
<br />
>phsC <br/><br />
Atgaatactatctggggagcggaactacattatgcgccagattattggccgctgtggttaa<br />
tttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcg<br />
ccggatgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctg<br />
gcgattcgccgctggcattggggaaatgcgttactgtttgttttattactgttaagcggtt<br />
tatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtgg<br />
ttttgccttactggctttctgggtcgggtttgtgctgatcaacctcaccacaggtaacggg<br />
cgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgttttt<br />
acctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagtt<br />
caatccactgcaacaactggcatatctggcgattatgtacgcgctggtaccgctgttaatc<br />
atcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgc<br />
tgcatatggcgcttgctatcatcggcttactgtttatttgcgcgcatctctatctgtgtac<br />
tcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgtcatcgt<br />
accgcgccgcgcggggataagtccgccgtctga <br/><br />
<br />
<br />
<!------------- sequence data-------------><br />
</div><br />
<br/><br />
<br />
<br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 6/16--Checked on spotted cell survival assay, collected MOPS minimal media materials & started making component solutions<br />
<br />
</li><br />
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<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<b> Minimal Medium Work </b> <br/><br />
In Jay Keasling's work with thiosulfate reductase he used a variation of MOPS minimal medium designed to avoid background metal presence from interfering with measurement of bacterial metal removal potential. Began making the various necessary component solutions as detailed in this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/><br />
<b>Survival of bacteria in copper growth assay </b> <br/><br />
Previously had plated a drop of each culture from the narrow concentration range copper growth assay on 6/14 to see if the cultures that had stopped growing/never grew were still alive. Observed the following results, where g signifies growth and - signifies no growth:<br/><br />
<table><br />
<tr><br />
<td>cell line</td><br />
<td>0 M Cu<sup>2+</sup></td><br />
<td>500 uM Cu<sup>2+</sup></td><br />
<td>600 uM Cu<sup>2+</sup></td><br />
<td>700 uM Cu<sup>2+</sup></td><br />
<td>800 uM Cu<sup>2+</sup></td><br />
<td>900 uM Cu<sup>2+</sup></td><br />
<td>1 mM Cu<sup>2+</sup></td><br />
<td>1.5 mM Cu<sup>2+</sup></td><br />
<td>2 mM Cu<sup>2+</sup></td><br />
<td>3 mM Cu<sup>2+</sup></td><br />
<td>4 mM Cu<sup>2+</sup></td><br />
<td>1 M Cu<sup>2+</sup></td><br />
</tr><br />
<tr><br />
<td>DH5alpha</td><br />
<td>- </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>- </td><br />
<td>g </td><br />
<td>- </td><br />
<td>- </td><br />
<td>g </td><br />
<td>g </td><br />
<td>- </td><br />
</tr><br />
<tr><br />
<td>LE392</td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>g </td><br />
<td>- </td><br />
</tr><br />
</table><br />
It is unsurprising that all cells should die at 1 M copper sulfate concentrations, but the lack of growth of DH5alpha at 0 M, 900 uM, etc. seems anomalous, given that the cells clearly survived at higher copper levels and may reflect an insufficient amount of culture spotted in those cases. <br/><br />
<i>This information is also recorded on page 11 of the hard copy lab notebook. </i> <br/><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 6/17--more minimal media work and meeting, started BL21 culture<br />
<br />
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<br />
<!------------- Thursday -------------><br />
<b>Modified MOPS minimal medium</b><br/><br />
Finished creation of a stock solution according the this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/><br />
<b>Additional <i> E. coli </i> strain </b> <br/><br />
Began a culture of BL21 as another hardy option for future assays. <br/><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 6/18--Arrival of plasmid pSB74, transformation of Biobricks, & copper growth assays for BL21 strain. <br />
<br />
</li><br />
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<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<ul><br />
<li>Plasmid pSB74, which contains the thiosulfate reductase gene, arrived from Addgene, already in some unspecified <i>E coli</i>, so plated them out on an ampicillin LB plate and put in incubator at 37.</li> <br />
<li>Transformed constitutive promote J23114, IPTG-inducible promoter R0011, terminator B0015, and repressor C0012(for inducible promoter) into BL21 according to standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation protocol </a> and plated onto ampicillin LB plates</li><br />
<li>Also ran BL21 copper growth assays for wide and narrow concentrations ranges according to the modified protocol used on 6/14, starting with dilution of a culture of OD 0.873.</li><br />
</ul><br />
<i> The activities of this day are also recorded on pages 13-15 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<li><br />
<br />
Saturday 6/19--Redo of Biobrick transformations & analysis of BL21 copper growth assay<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a><br />
<div style="display:none;" id="sunday"><br />
<!------------- Saturday -------------><br />
<b>Transformation troubles</b> <br/><br />
While the pSB74 culture grew, none of the BL21 transformants from 6/19 did, so repeated the transformations of Biobricks B0015, R0011, C0012,and J23114 into LE392 cells, once again following this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation protocol</a> and plating onto ampicillin LB plates. The remaining transformant solutions were set aside in case of another failure.<br/><br />
<b>BL21 copper growth assay analysis</b> <br/><br />
Retrieved the following data regarding BL21's growth in copper solution: <br/><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/f/f6/Yale-bl21.jpg" /><br />
</div><br />
It appears that BL21 is slightly more sensitive to copper than the other two strains, since 3 mM levels of copper(II) sulfate are enough to almost completely inhibit BL21's growth. <br/><br />
<i> The activities of this day are also recorded on page 15 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Sunday 6/20--Observed growth of all transformants from 6/19, so used them and the culture containing pSB74 to inoculate 5 mL liquid cultures in LB with ampicillin. Left to grow overnight on shaker at 37˚C for miniprep the following morning. <br/><br />
<i> The activities of this day are also recorded on page 16 of the hard copy lab notebook </i><br />
<br />
</li><br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
</ul><br />
</p><br />
</div><br />
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<br />
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</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_8Team:Yale/Our Project/Notebook/Week 82010-10-27T20:55:14Z<p>Litagaki: </p>
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<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
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<h5><br />
lab notebook: week 8 (7/26-8/1)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/26--<br />
<br />
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<!------------- Monday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 82 of the lab notebook hard copy. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/27--<br />
</li><br />
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<br />
<br />
<!------------- Tuesday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 83 of the lab notebook hard copy. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/28<br />
<br />
</li><br />
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<!------------- Wednesday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 83 & 84 of the lab notebook hard copy. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/29--<br />
<br />
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<!------------- Thursday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 84 of the lab notebook hard copy. </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/30--<br />
<br />
</li><br />
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<!------------- Friday -------------><br />
Extra content<br />
<i> This day's work is also recorded on page 85 of the lab notebook hard copy. </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday short content<br />
<br />
</li><br />
<a id="link" href="#" onclick="toggle_visibility('saturday'); return false;">See more</a><br />
<div style="display:none" id="saturday"><br />
<!------------- Saturday -------------><br />
Extra content<br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday short content<br />
<br />
</li><br />
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<div style="display:none" id="sunday"><br />
<!------------- Sunday -------------><br />
Extra content<br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-27T20:41:51Z<p>Litagaki: </p>
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<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
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<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/19--Investigation of how pre-ligation processing affects ligation results<br />
<br />
</li><br />
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<!------------- Monday -------------><br />
<ul><br />
<b>Gel Purification of PCR product </b> <br/><br />
<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract " gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li><br />
<b>Self-ligation of phsABC to test restriction enzymes </b> <br/><br />
<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li><br />
<b> Dual-purpose gel </b> <br/><br />
<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li><br />
<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li><br />
<b>Double digest of gel-purified phsABC </b> <br/><br />
<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li><br />
<b>AvaII digestions to check for inhibition </b> <br/><br />
<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li><br />
<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li><br />
<b>Separation of phsABC double digestion </b> <br/><br />
<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.<br />
Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li><br />
<br />
</ul><br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
Extra content<br />
<br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
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</div><br />
<br />
<li><br />
<br />
Tuesday 7/20--<br />
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<!------------- Tuesday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--<br />
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</li><br />
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<!------------- Wednesday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--<br />
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</li><br />
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<!------------- Thursday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--<br />
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<!------------- Friday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--<br />
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<!------------- Saturday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
</li><br />
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<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
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<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_7Team:Yale/Our Project/Notebook/Week 72010-10-27T19:41:10Z<p>Litagaki: </p>
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<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
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<div id="right-col"><br />
<h5><br />
lab notebook: week 7 (7/19-7/25)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
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<li><br />
<br />
Monday 7/19--<br />
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<!------------- Monday -------------><br />
Extra content<br />
<br />
<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
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<li><br />
<br />
Tuesday 7/20--<br />
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<br />
<!------------- Tuesday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/21--<br />
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<!------------- Wednesday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 76-78 of the hard copy lab notebook </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/22--<br />
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<!------------- Thursday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 79 of the hard copy lab notebook </i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/23--<br />
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<!------------- Friday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook </i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/24--<br />
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<!------------- Saturday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/25--<br />
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<!------------- Sunday -------------><br />
Extra content<br />
<i>This day's labwork is also recorded on page 81 of the hard copy lab notebook </i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T19:32:18Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
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<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
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<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. Restarted power source, let run, and then visualized the gels </li><br />
<li>Digestion of miniprep shows that all ligation efforts failed--had they succeeded there would have been fragments at 7.8 kb, but as the gel below shows, all the samples run at slightly over 3 kb (ladder rungs are 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 8 kb, & 10 kb).</li><br />
<br />
<li> Efforts to visualize the colony PCR gel failed entirely--maybe forgot to add ethidium bromide? But given above results, it's a moot point. </li><br />
</ul><br />
<h4> Copper Removal Assay Prep </h4><br />
<ul><br />
<li> Want to run an assay measuring whether pSB74 transformants remove copper(II) from their surroundings. If had a large bacterial culture with an intermediate CuSO<sub>4</sub> concentration (say 500 uM), could periodically remove small aliquots, centrifuge out the cells, and spectrophotometrically determine the copper content. </li><br />
<li> Need to determine what wavelength should be used to monitor copper concentration and create a calibration curve relating absorbance at that wavelength to copper concentration. </li><br />
<li> Start by making serial dilutions of CuSO<sub>4</sub> in LB. Made samples of 0.1 M, 10 mM, 1 mM, 100 uM, 10 uM, 1 uM and 0.1 uM concentrations. </li><br />
<li> While spectrum had no clear peak, the CuSO<sub>4</sub> did absorb strongly at 700 nm compared to just LB, so chose that as wavelength to monitor. Attempted to establish curve, but got the odd result that the 10 uM absorbed orders of magnitude more than the 100 uM. Will redo dilutions and try again another day. </li><br />
<li> Inoculated 5 mL liquid cultures of LE392, both with and without pSB74, adding 5 uL of 1000x ampicillin to the transformant culture. Let grow overnight on shaker at 37˚C. </li><br />
<br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015<br />
<br />
</li><br />
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<br />
<br />
<!------------- Wednesday -------------><br />
<h4> Digest/Ligation Attempt #5 </h4><br />
<ul><br />
<li> Concerned that ligation components may not be thoroughly digested, so ran an all day digestion of phsABC, which containted 5 uL EcoRI buffer, 0.5 uL 100x BSA, 36.1 uL phsABC (at 27.7 ng/uL for 1 ug total), 1.8 uL EcoRI, 1.8 uL SpeI, and 4.8 uL water. </li><br />
<li> Also concerned about SpeI activity, so ran the following diagnostic SpeI digestion of B0015 that will then be run on a gel versus the circularized plasmid: 5 uL NEB buffer 4, 0.5 uL 100x BSA, 4 uL B0015(1 ug DNA), 3.6 uL SpeI, and 36.9 uL water, let to run for 2 hours at 37˚C before heat killing at 80˚C for twenty minutes </li><br />
<li>Simultaneously digested more B0015 with XbaI according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5">7/8 </a> to ensure there will be enough vector. </li><br />
<li> Purified the XbaI-digested B0015 with a standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>, eluting in 40 uL, and then digested all of the result with 3.6 uL EcoRI, 5 uL EcoRI buffer, 0.5 uL 100x BSA, and 0.9 uL of water, letting it run for two hours. An hour into the digestion, added 1 uL of CIP to the reaction, and after the digestion heat-killed the enzymes with 20 minutes at 80˚C. </li><br />
</ul><br />
<h4>Copper(II) Absorbance Calibration Curve Redo</h4><br />
<ul><br />
<li> Once again carefully make serial dilutions of copper sulfate in LB, creating a series of solutions running from 0.1 M to 1 uM and separated by a power of ten. Absorbances prove to be highly nonlinear again, so research and find that copper(II) concentration cannot directly be measured directly spectrophotometrically. </li><br />
</ul><br />
<h4> Bacterial Survival in Copper Solution </h4><br />
<ul><br />
<li> Retrieve from incubator plates spotted with copper solution cultures from 6/13 copper growth assay. Find that all cultures up to and including 4 mM copper levels survived, both in the transformed and untransformed LE392. Also see a colony were the pSB74 transformant in 50 mM copper was spotted, but as there is no growth at 10 mM, wonder if there was an accidental drip during spotting. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--Confirmation of SpeI activity and EtOH precipitation of ligation components<br />
<br />
</li><br />
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<!------------- Thursday -------------><br />
<ul><br />
<b>Analysis of SpeI activity</b> <br/><br />
<li> Ran the SpeI digest of B0015 on a gel versus the circular B0015 and a 1 kb ladder (leftmost lane). The digested plasmid (rightmost lane) ran slower than the undigested one (middle lane), confirming that SpeI is active and successfully linearized the plasmid. </li><br />
</ul><br />
<b>Ethanol precipitation of ligation components </b> <br/><br />
<ul><br />
<li> Guessing that higher concentrations of insert and vector might improve ligation results, ethanol precipitate the digested samples from 7/14 according to the following <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> protocol </a> </li><br />
<li> In case need more material, started five overnight liquid cultures of each pSB74 and B0015 in Amp LB for morning miniprep. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on page 69 of the hard copy lab notebook.</i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--Experimentation with different treatments of vector & insert DNA prior to ligation<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
<h4>Stockpiling starting materials </h4><br />
<ul><br />
<li> Miniprepped the overnight cultures of pSB74 and B0015 <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> by vacuum manifold </a> and found the resulting concentrations by nanodrop. The five B0015 samples had concentrations of 83.7, 83.4, 111.7, 130.0, and 103.3 ng/uL while the pSB74 samples had concentration values of 118.7, 129.5, 118.2, 137.3, and 124.9 ng/uL. </li><br />
<li> Set up 8 PCR reactions with pSB74 samples using the "phs50" thermocycler protocol and the DMSO variant of PCR reaction contents from <a href=" https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4"> 6/30 </a>.</li><br />
<li> Also ran four digestions of B0015 with XbaI, each of which included 5 uL NEB buffer 4, 0.5 uL 100x BSA, 3.6 uL XbaI, 7.7 uL B0015 (1 ug worth), and 33.2 uL of water. Let these run for two hours at 37˚C </li><br />
<li> Meanwhile attempted to resuspend ethanol precipitated DNA, but nanodrop showed no DNA present--pellets must have fallen out of tubes when inverted to dry. </li><br />
<li> After XbaI digestion of B0015 skipped PCR purification step (concerned that it was leading to product loss)and simply added 0.5 uL of 5 M NaCl to each digestion so that the buffer solution would have the salt content required by EcoRI. Then added 2 uL of EcoRI to each digestion and let incubate for 2 hours at 37˚C. One hour into this digestion, added 1 uL of CIP to half (two) of the digestion reactions (want to see if it makes a difference). </li><br />
<li> Following the PCR amplification of phsABC, ran the vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR cleanup protocol</a> on four out of the eight reaction solutions and measured the DNA concentrations of the resulting solutions as 99.5, 131.2, 75.5, 36.2, and 26.2 ng/uL. </li><br />
<li> Concerned that phsABC may not be getting digested properly, especially given that its cut sites are so near its ends. While running the required EcoRI/SpeI double digestion, will run in parallel single digestions of phsABC with each EcoRI and SpeI. While the amount cut off makes these digestions impossible to detect directly, if the digestions occur properly the resulting fragments should be able to self-ligate, so it is possible to test for that. </li><br />
<li> The two double digestions had contents as follows: 5 uL EcoRI buffer, 0.5 uL 100x BSA, 10.5 uL phsABC (1 ug DNA), 1.8 uL EcoRI, 1.8 uL SpeI, and 30.4 uL water. </li><br />
<li> The diagnostic single digestions were run with 13.2 uL phsABC (at 75.5 ng/uL, 1 ug), 5 uL EcoRI buffer, 0.5 uL BSA, 27.7 uL water, and 3.6 uL of the relevant enzyme whether EcoRI or SpeI. </li><br />
<li> All of the above digestions were run for eight hours at 37˚C before being heat-killed with 20 minutes at 80˚C. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 70 & 71 of the hard copy lab notebook.</i><br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/ProtocolsTeam:Yale/Our Project/Protocols2010-10-27T19:02:23Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></b></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
protocols<br />
</h5><br />
<p><br />
<!------------- PROTOCOL: NEEDS TO BE EDITED-------------><br />
<h4> Bacterial protocols </h4> <br />
<br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation</a><br />
<br />
<h4> DNA protocols </h4> <br />
<br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep">miniprep</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">gel extraction</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate">ethanol precipitation</a> <br />
<br />
<br />
<h4> Recipes </h4> <br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">MOPS minimal medium</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/SOC_broth">SOC medium</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/LB_agar">LB agar</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/TSI_agar">Triple Sugar Iron (TSI) agar</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/bacteria mixture for H2S Assay">Bacteria Mixture for H<sub>2</sub>S Assay</a><br/><br />
<br />
<h4> Copper absorbance protocols </h4><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/standard_curve">standard absorbance curve measurement</a> <br/><br />
<a id="link" href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/disappearance_curve">copper disappearance as a function of bacterial growth curve</a> <br/><br />
<br />
<!------------- PROTOCOL: NEEDS TO BE EDITED -------------><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T19:01:23Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. Restarted power source, let run, and then visualized the gels </li><br />
<li>Digestion of miniprep shows that all ligation efforts failed--had they succeeded there would have been fragments at 7.8 kb, but as the gel below shows, all the samples run at slightly over 3 kb (ladder rungs are 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 8 kb, & 10 kb).</li><br />
<br />
<li> Efforts to visualize the colony PCR gel failed entirely--maybe forgot to add ethidium bromide? But given above results, it's a moot point. </li><br />
</ul><br />
<h4> Copper Removal Assay Prep </h4><br />
<ul><br />
<li> Want to run an assay measuring whether pSB74 transformants remove copper(II) from their surroundings. If had a large bacterial culture with an intermediate CuSO<sub>4</sub> concentration (say 500 uM), could periodically remove small aliquots, centrifuge out the cells, and spectrophotometrically determine the copper content. </li><br />
<li> Need to determine what wavelength should be used to monitor copper concentration and create a calibration curve relating absorbance at that wavelength to copper concentration. </li><br />
<li> Start by making serial dilutions of CuSO<sub>4</sub> in LB. Made samples of 0.1 M, 10 mM, 1 mM, 100 uM, 10 uM, 1 uM and 0.1 uM concentrations. </li><br />
<li> While spectrum had no clear peak, the CuSO<sub>4</sub> did absorb strongly at 700 nm compared to just LB, so chose that as wavelength to monitor. Attempted to establish curve, but got the odd result that the 10 uM absorbed orders of magnitude more than the 100 uM. Will redo dilutions and try again another day. </li><br />
<li> Inoculated 5 mL liquid cultures of LE392, both with and without pSB74, adding 5 uL of 1000x ampicillin to the transformant culture. Let grow overnight on shaker at 37˚C. </li><br />
<br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
<h4> Digest/Ligation Attempt #5 </h4><br />
<ul><br />
<li> Concerned that ligation components may not be thoroughly digested, so ran an all day digestion of phsABC, which containted 5 uL EcoRI buffer, 0.5 uL 100x BSA, 36.1 uL phsABC (at 27.7 ng/uL for 1 ug total), 1.8 uL EcoRI, 1.8 uL SpeI, and 4.8 uL water. </li><br />
<li> Also concerned about SpeI activity, so ran the following diagnostic SpeI digestion of B0015 that will then be run on a gel versus the circularized plasmid: 5 uL NEB buffer 4, 0.5 uL 100x BSA, 4 uL B0015(1 ug DNA), 3.6 uL SpeI, and 36.9 uL water, let to run for 2 hours at 37˚C before heat killing at 80˚C for twenty minutes </li><br />
<li>Simultaneously digested more B0015 with XbaI according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5">7/8 </a> to ensure there will be enough vector. </li><br />
<li> Purified the XbaI-digested B0015 with a standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>, eluting in 40 uL, and then digested all of the result with 3.6 uL EcoRI, 5 uL EcoRI buffer, 0.5 uL 100x BSA, and 0.9 uL of water, letting it run for two hours. An hour into the digestion, added 1 uL of CIP to the reaction, and after the digestion heat-killed the enzymes with 20 minutes at 80˚C. </li><br />
</ul><br />
<h4>Copper(II) Absorbance Calibration Curve Redo</h4><br />
<ul><br />
<li> Once again carefully make serial dilutions of copper sulfate in LB, creating a series of solutions running from 0.1 M to 1 uM and separated by a power of ten. Absorbances prove to be highly nonlinear again, so research and find that copper(II) concentration cannot directly be measured directly spectrophotometrically. </li><br />
</ul><br />
<h4> Bacterial Survival in Copper Solution </h4><br />
<ul><br />
<li> Retrieve from incubator plates spotted with copper solution cultures from 6/13 copper growth assay. Find that all cultures up to and including 4 mM copper levels survived, both in the transformed and untransformed LE392. Also see a colony were the pSB74 transformant in 50 mM copper was spotted, but as there is no growth at 10 mM, wonder if there was an accidental drip during spotting. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--Confirmation of SpeI activity and EtOH precipitation of ligation components<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
<ul><br />
<b> Analysis of SpeI activity</b> <br/><br />
<li> Ran the SpeI digest of B0015 on a gel versus the circular B0015 and a 1 kb ladder (leftmost lane). The digested plasmid (rightmost lane) ran slower than the undigested one (middle lane), confirming that SpeI is active and successfully linearized the plasmid. </li><br />
</ul><br />
<b>Ethanol precipitation of ligation components </b> <br/><br />
<ul><br />
<li> Guessing that higher concentrations of insert and vector might improve ligation results, ethanol precipitate the digested samples from 7/14 according to the following <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/EtOH_precipitate"> protocol </a> </li><br />
<i> Wetlab work for this day is also recorded on page 69 of the hard copy lab notebook.</i><br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T18:51:40Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. Restarted power source, let run, and then visualized the gels </li><br />
<li>Digestion of miniprep shows that all ligation efforts failed--had they succeeded there would have been fragments at 7.8 kb, but as the gel below shows, all the samples run at slightly over 3 kb (ladder rungs are 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 8 kb, & 10 kb).</li><br />
<br />
<li> Efforts to visualize the colony PCR gel failed entirely--maybe forgot to add ethidium bromide? But given above results, it's a moot point. </li><br />
</ul><br />
<h4> Copper Removal Assay Prep </h4><br />
<ul><br />
<li> Want to run an assay measuring whether pSB74 transformants remove copper(II) from their surroundings. If had a large bacterial culture with an intermediate CuSO<sub>4</sub> concentration (say 500 uM), could periodically remove small aliquots, centrifuge out the cells, and spectrophotometrically determine the copper content. </li><br />
<li> Need to determine what wavelength should be used to monitor copper concentration and create a calibration curve relating absorbance at that wavelength to copper concentration. </li><br />
<li> Start by making serial dilutions of CuSO<sub>4</sub> in LB. Made samples of 0.1 M, 10 mM, 1 mM, 100 uM, 10 uM, 1 uM and 0.1 uM concentrations. </li><br />
<li> While spectrum had no clear peak, the CuSO<sub>4</sub> did absorb strongly at 700 nm compared to just LB, so chose that as wavelength to monitor. Attempted to establish curve, but got the odd result that the 10 uM absorbed orders of magnitude more than the 100 uM. Will redo dilutions and try again another day. </li><br />
<li> Inoculated 5 mL liquid cultures of LE392, both with and without pSB74, adding 5 uL of 1000x ampicillin to the transformant culture. Let grow overnight on shaker at 37˚C. </li><br />
<br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
<h4> Digest/Ligation Attempt #5 </h4><br />
<ul><br />
<li> Concerned that ligation components may not be thoroughly digested, so ran an all day digestion of phsABC, which containted 5 uL EcoRI buffer, 0.5 uL 100x BSA, 36.1 uL phsABC (at 27.7 ng/uL for 1 ug total), 1.8 uL EcoRI, 1.8 uL SpeI, and 4.8 uL water. </li><br />
<li> Also concerned about SpeI activity, so ran the following diagnostic SpeI digestion of B0015 that will then be run on a gel versus the circularized plasmid: 5 uL NEB buffer 4, 0.5 uL 100x BSA, 4 uL B0015(1 ug DNA), 3.6 uL SpeI, and 36.9 uL water, let to run for 2 hours at 37˚C before heat killing at 80˚C for twenty minutes </li><br />
<li>Simultaneously digested more B0015 with XbaI according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5">7/8 </a> to ensure there will be enough vector. </li><br />
<li> Purified the XbaI-digested B0015 with a standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>, eluting in 40 uL, and then digested all of the result with 3.6 uL EcoRI, 5 uL EcoRI buffer, 0.5 uL 100x BSA, and 0.9 uL of water, letting it run for two hours. An hour into the digestion, added 1 uL of CIP to the reaction, and after the digestion heat-killed the enzymes with 20 minutes at 80˚C. </li><br />
</ul><br />
<h4>Copper(II) Absorbance Calibration Curve Redo</h4><br />
<ul><br />
<li> Once again carefully make serial dilutions of copper sulfate in LB, creating a series of solutions running from 0.1 M to 1 uM and separated by a power of ten. Absorbances prove to be highly nonlinear again, so research and find that copper(II) concentration cannot directly be measured directly spectrophotometrically. </li><br />
</ul><br />
<h4> Bacterial Survival in Copper Solution </h4><br />
<ul><br />
<li> Retrieve from incubator plates spotted with copper solution cultures from 6/13 copper growth assay. Find that all cultures up to and including 4 mM copper levels survived, both in the transformed and untransformed LE392. Also see a colony were the pSB74 transformant in 50 mM copper was spotted, but as there is no growth at 10 mM, wonder if there was an accidental drip during spotting. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T18:49:54Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. Restarted power source, let run, and then visualized the gels </li><br />
<li>Digestion of miniprep shows that all ligation efforts failed--had they succeeded there would have been fragments at 7.8 kb, but as the gel below shows, all the samples run at slightly over 3 kb (ladder rungs are 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 8 kb, & 10 kb).</li><br />
<br />
<li> Efforts to visualize the colony PCR gel failed entirely--maybe forgot to add ethidium bromide? But given above results, it's a moot point. </li><br />
</ul><br />
<h4> Copper Removal Assay Prep </h4><br />
<ul><br />
<li> Want to run an assay measuring whether pSB74 transformants remove copper(II) from their surroundings. If had a large bacterial culture with an intermediate CuSO<sub>4</sub> concentration (say 500 uM), could periodically remove small aliquots, centrifuge out the cells, and spectrophotometrically determine the copper content. </li><br />
<li> Need to determine what wavelength should be used to monitor copper concentration and create a calibration curve relating absorbance at that wavelength to copper concentration. </li><br />
<li> Start by making serial dilutions of CuSO<sub>4</sub> in LB. Made samples of 0.1 M, 10 mM, 1 mM, 100 uM, 10 uM, 1 uM and 0.1 uM concentrations. </li><br />
<li> While spectrum had no clear peak, the CuSO<sub>4</sub> did absorb strongly at 700 nm compared to just LB, so chose that as wavelength to monitor. Attempted to establish curve, but got the odd result that the 10 uM absorbed orders of magnitude more than the 100 uM. Will redo dilutions and try again another day. </li><br />
<li> Inoculated 5 mL liquid cultures of LE392, both with and without pSB74, adding 5 uL of 1000x ampicillin to the transformant culture. Let grow overnight on shaker at 37˚C. </li><br />
<br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--Copper Removal assay work and 5th attempt to ligate phsABC into terminator B0015<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
<h4> Digest/Ligation Attempt #5 </h4><br />
<ul><br />
<li> Concerned that ligation components may not be thoroughly digested, so ran an all day digestion of phsABC, which containted 5 uL EcoRI buffer, 0.5 uL 100x BSA, 36.1 uL phsABC (at 27.7 ng/uL for 1 ug total), 1.8 uL EcoRI, 1.8 uL SpeI, and 4.8 uL water. </li><br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</li><br />
<li> Also concerned about SpeI activity, so ran the following diagnostic SpeI digestion of B0015 that will then be run on a gel versus the circularized plasmid: 5 uL NEB buffer 4, 0.5 uL 100x BSA, 4 uL B0015(1 ug DNA), 3.6 uL SpeI, and 36.9 uL water, let to run for 2 hours at 37˚C before heat killing at 80˚C for twenty minutes </li><br />
<li>Simultaneously digested more B0015 with XbaI according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5">7/8 </a> to ensure there will be enough vector. </li><br />
<li> Purified the XbaI-digested B0015 with a standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>, eluting in 40 uL, and then digested all of the result with 3.6 uL EcoRI, 5 uL EcoRI buffer, 0.5 uL 100x BSA, and 0.9 uL of water, letting it run for two hours. An hour into the digestion, added 1 uL of CIP to the reaction, and after the digestion heat-killed the enzymes with 20 minutes at 80˚C. </li><br />
</ul><br />
<h4>Copper(II) Absorbance Calibration Curve Redo</h4><br />
<ul><br />
<li> Once again carefully make serial dilutions of copper sulfate in LB, creating a series of solutions running from 0.1 M to 1 uM and separated by a power of ten. Absorbances prove to be highly nonlinear again, so research and find that copper(II) concentration cannot directly be measured directly spectrophotometrically. </li><br />
</ul><br />
<h4> Bacterial Survival in Copper Solution </h4><br />
<ul><br />
<li> Retrieve from incubator plates spotted with copper solution cultures from 6/13 copper growth assay. Find that all cultures up to and including 4 mM copper levels survived, both in the transformed and untransformed LE392. Also see a colony were the pSB74 transformant in 50 mM copper was spotted, but as there is no growth at 10 mM, wonder if there was an accidental drip during spotting. </li><br />
</ul><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T18:24:40Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. Restarted power source, let run, and then visualized the gels </li><br />
<li>Digestion of miniprep shows that all ligation efforts failed--had they succeeded there would have been fragments at 7.8 kb, but as the gel below shows, all the samples run at slightly over 3 kb (ladder rungs are 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 8 kb, & 10 kb).</li><br />
<br />
<li> Efforts to visualize the colony PCR gel failed entirely--maybe forgot to add ethidium bromide? But given above results, it's a moot point. </li><br />
</ul><br />
<h4> Copper Removal Assay Prep </h4><br />
<ul><br />
<li> Want to run an assay measuring whether pSB74 transformants remove copper(II) from their surroundings. If had a large bacterial culture with an intermediate CuSO<sub>4</sub> concentration (say 500 uM), could periodically remove small aliquots, centrifuge out the cells, and spectrophotometrically determine the copper content. </li><br />
<li> Need to determine what wavelength should be used to monitor copper concentration and create a calibration curve relating absorbance at that wavelength to copper concentration. </li><br />
<li> Start by making serial dilutions of CuSO<sub>4</sub> in LB. Made samples of 0.1 M, 10 mM, 1 mM, 100 uM, 10 uM, 1 uM and 0.1 uM concentrations. </li><br />
<li> While spectrum had no clear peak, the CuSO<sub>4</sub> did absorb strongly at 700 nm compared to just LB, so chose that as wavelength to monitor. Attempted to establish curve, but got the odd result that the 10 uM absorbed orders of magnitude more than the 100 uM. Will redo dilutions and try again another day. </li><br />
<li> Inoculated 5 mL liquid cultures of LE392, both with and without pSB74, adding 5 uL of 1000x ampicillin to the transformant culture. Let grow overnight on shaker at 37˚C. </li><br />
<br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
Extra content<br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T17:38:23Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<h4> Ligation attempt #4 results</h4><br />
<ul><br />
<li> Ran 1.0% agarose gels of both the colony PCR reactions and EcoRI digestion of minipreps. Gels contained 10 uL of ethidium bromide and were run at 90 V with a 1 kb ladder, but the power source malfunctioned and turned off at some point, so the gel sat for an unknown amount of time. </li><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
Extra content<br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T17:34:34Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> pSB74 transformant copper growth assay</h4><br />
<ul><br />
<li> Looked at results of the previous day's assay and saw that the presence of pSB74 actually decreased the copper tolerance of the LE392. Maybe the effort associated with protein production actually weakens the cell's ability to deal with the copper? </li><br />
<li> Spotted each culture from the 96-well plate onto an agar plate (LB and ampicillin in the case of transformants, plain LB in the case of the untransformed) and put in incubator to see if it grows. </li><br />
</ul><br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
Extra content<br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
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<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T17:24:13Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.</li><br />
<li> Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL</li><br />
<li> Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water. </li><br />
<b> Digest of miniprepped attempt #4 products </b> <br/><br />
<li> Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size.<br />
Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.</li><br />
<b> Colony PCR of ligation attempt #4 transformants </b> <br/><br />
<li> Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol</li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
Extra <br />
<i> Wetlab work for this day is also recorded on pages 65-67 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
Extra content<br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6Team:Yale/Our Project/Notebook/Week 62010-10-27T16:48:44Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 6 (7/12-7/18)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<br />
<!------------- Monday -------------><br />
<h4> Copper Growth Assay Comparing LE392 With And Without pSB74</h4><br />
<ul><br />
<li>Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:<br/><br />
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase. </li><br />
<li> Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75. </li><br />
<li> Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> before. </a></li> <br/><br />
Row A: Wide copper concentration range blank series with Amp & IPTG <br/><br />
Row B: Narrow copper concentration range blank series with Amp & IPTG <br/><br />
Row C: Wide copper concentration range transformant series with Amp & IPTG <br/><br />
Row D: Narrow copper concentration range transformant series with Amp & IPTG <br/><br />
Row E: Wide copper concentration range blank series<br/><br />
Row F: Narrow copper concentration range blank series<br/><br />
Row G: Wide copper concentration range untransformed series<br/><br />
Row H: Narrow copper concentration range untransformed series<br/><br />
</ul><br />
<h4> Continuing ligation work </h4><br />
<ul><br />
<b>Miniprep and sequencing of attempt #4 transformants </b><br/><br />
<li>Using the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> vacuum manifold protocol </a> miniprepped the cultures grown up from the sixteen colonies (#9-#24) that<br />
<i>Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.</i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/13--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
Extra <br />
<i> Wetlab work for this day is also recorded on pages 65-66 of the hard copy lab notebook.</i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/14--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<br />
<!------------- Wednesday -------------><br />
Extra content<br />
<i> Wetlab work for this day is also recorded on pages 67-68 of the hard copy lab notebook.</i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/15--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('thursday')">See more/less</a><br />
<div style="display:none;" id="thursday"><br />
<!------------- Thursday -------------><br />
Extra content<br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/16--<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('friday')">See more/less</a><br />
<div style="display:none;" id="friday"><br />
<!------------- Friday -------------><br />
Extra content<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
</p><br />
</div><br />
<br />
<br />
</div><br />
</body><br />
</html></div>Litagakihttp://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_5Team:Yale/Our Project/Notebook/Week 52010-10-27T16:48:16Z<p>Litagaki: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Yale/Templates/Yale_Header_Project}}<br />
<html><br />
<body><br />
<div id="main-content"><br />
<div id="left-col"><br />
<h5><br />
our project<br />
</h5><br />
<p><br />
<ul id="proj-nav"><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project">introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Applications">applications</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Methods">methods</a></li><br />
<li><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">lab notebook</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook">week 1</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 2">week 2</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 3">week 3</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 4">week 4</a></li><br />
<li id="nb"><b><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 5">week 5</a></b></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 6">week 6</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li><br />
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li><br />
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Future Work">future work</a></li><br />
</ul><br />
</p><br />
</div><br />
<br />
<div id="right-col"><br />
<h5><br />
lab notebook: week 5 (7/5 -7/11)<br />
</h5><br />
<p><br />
<ul><br />
<!------------- LAB NOTEBOOK -------------><br />
<br />
<li><br />
<br />
Monday 7/5--Colony PCR of Ligation Attempt #2 Transformants<br />
<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('monday')">See more/less</a><br />
<div style="display:none;" id="monday"><br />
<br />
<!------------- Monday -------------><br />
<h4> Colony PCR</h4><br />
<ul><br />
<li>Ran colony PCR of colonies 1B-9B from ligation attempt #2 according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4"> 7/1</a>.</li><br />
<li>Then ran a 1.0% agarose gel of the nine PCR reaction solutions against a 1 kb ladder, but the resulting gel failed to show any evidence of either insert or vector, so will have to rely on sequencing and digests of miniprepped plasmids.</li><br />
Gel of colony PCR from ligation attempt #2 Lane 1: 1 kb ladder, Lanes 3-11:PCR products from colonies 1B-9B, in order<br/><br />
<br />
<li>Miniprepped liquid cultures grown from colonies of first ligation attempt (colonies 1A-4A) and second ligation attempt (colonies 1B-9B) according to <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard miniprep protocol.</a> </li><br />
</ul><br />
<i>This day's activities are also recorded on page 47 and 48 of the hard copy lab notebook. </i><br />
<!------------- Monday -------------><br />
<br />
</div><br />
<br />
<li><br />
<br />
Tuesday 7/6--Analysis of Results of Ligation Attempts 1 & 2<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('tuesday')">See more/less</a><br />
<div style="display:none;" id="tuesday"><br />
<br />
<!------------- Tuesday -------------><br />
<h4> More Diagnostics of Ligation Results</h4><br />
<ul><br />
<li>In order to determine success of ligation attempts 1 and 2, digested the miniprepped plasmids 1A-4A and 1B-9B from 7/5 with XbaI and SpeI and then looked for evidence of two fragments, one at 3.2 kb and the other at 3.6 kb. Reactions were conducted for 2 hours at 37°C, with the following contents for each of the thirteen reactions </li><br />
<table><br />
<tr><td><br />
Component </td> <td> Volume<br />
</td> </tr><br />
<tr> <td><br />
Distilled Water </td> <td>6.4 μL<br />
</td> </tr><br />
<tr> <td><br />
10x NEB Buffer 4 </td> <td>2 μL<br />
</td> </tr><br />
<tr> <td><br />
DNA solution </td> <td>10 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB XbaI </td> <td>0.8 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB SpeI </td> <td>0.8 μL<br />
</td> </tr><br />
<tr> <td><br />
Total </td> <td>20 μL<br />
</td></tr><br />
</table><br />
<br />
<li>The diagnostic 1.0% agarose gel was run at 90 V vs. a 1 kb ladder. Each of the thirteen samples gave one diffuse band around 3 kb and no evidence of the 3.6 kb phsABC fragment, so both ligation attempts #1 and #2 were unsuccessful.</li><br />
Gel of XbaI and SpeI double digest of plasmids from ligation attempts #1 and #2 Lane 1: 1 kb ladder, Lanes 2-5:1A-4A, Lanes 6-14: 1B-9B <br/><br />
<br />
<li>During digestion prepped plasmid samples for sequencing. Each sample contained 3 μL of plasmid soln (average concentration of 96 ng/μL), 2 μL of 4 mM VF2 (forward primer), and 13 μL of distilled water. Intended to follow up with reverse sequencing of more promising samples, but gel results made it a moot point. </li><br />
</ul><br />
<br />
<i>Wetlab work for this day is also recorded on pages 47, 49, and 50 in the hard copy lab notebook. </i><br />
<!------------- Tuesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Wednesday 7/7--Triple ligation attempt--promoter + thiosulfate reductase + terminator<br />
<br />
</li><br />
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a><br />
<div style="display:none;" id="wednesday"><br />
<br />
<!------------- Wednesday -------------><br />
<h4>Ligation Strategy </h4><br />
<ul><br />
<li>Failure of previous two ligation efforts prompted rethinking of ligation strategy. Will attempt a one-step triple ligation that will link phsABC to B0015 and insert them both into a standard single-resistance iGEM background. phsABC will be digested with EcoRI and SpeI, B0015 with XbaI and PstI, and pSB1C3 with EcoRI and PstI, allowing for them all to be ligated together. </li><br />
</ul><br />
<br />
<b>Prep work </b> <br/><br />
<ul><br />
<li>Needed more phsABC, so ran five PCR reactions to amplify it from plasmid pSB74. Reaction mixture contents were the same as the DMSO variant used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4">6/30 </a>as was the thermocycler protocol (phs50). The results were then run on a gel</li><br />
Gel of PCR amplified phsABC Extreme right lane:1 kb ladder, all other lanes PCR reaction mixtures and their spillover<br />
<br />
<li>Needed chloramphenicol plates for pSB1C3-based constructs, so mixed 350 μL of 50 μg/μL chloramphenicol with an autoclaved solution of 7.5 g agar and 12.5 g LB in 500 mL H2O and poured plates. </li><br />
<br />
<b>Digestions</b> <br/><br />
<br />
<li>Ran all three digestion reactions for 30 minutes at 37 °C before heat killing for 20 minutes at 80 °C. Digestion mixtures had the following components: </li><br />
<table><br />
<tr> <td><br />
Digestion Reaction Components<br />
</td></tr> <tr> <td><br />
phsABC Digestion </td> <td> B0015 Digestion </td> <td>pSB1C3 Digestionhttps://2010.igem.org/Main_Page<br />
</td></tr> <tr> <td><br />
5 μL EcoRI Buffer 10x </td> <td>5 μL NEB Buffer 3 10x </td> <td> 5 μL EcoRI Buffer 10x<br />
</td></tr> <tr> <td><br />
0.5 μL BSA 100x </td> <td>0.5 μL BSA 100x </td> <td>0.5 μL BSA 100x <br />
</td> </tr> <tr> <td><br />
21.1 μL phsABC soln (1 μg DNA)</td> <td> 3.8 μL B0015 soln (1 μg DNA) </td> <td>15 μL phsABC soln (375 ng DNA)<br />
</td></tr> <tr> <td><br />
1.8 μL EcoRI & 1.8 μL SpeI </td> <td>1.8 μL XbaI & 1.8 μL PstI </td> <td>1.2 μL EcoRI, 1.2 μL PstI, & 1.2 μL DpnI<br />
</td></tr> <tr> <td><br />
20.8 μL H2O </td> <td>37.1 μL H2O </td> <td> 15.9 μL H2O<br />
</td><br />
</tr><br />
</table><br />
</ul><br />
<i>Wetlab work for this day is also recorded on page 51 of the hard copy lab notebook. </i><br />
<!------------- Wednesday -------------><br />
</div><br />
<br />
<li><br />
<br />
Thursday 7/8--Further ligation efforts<br />
<br />
</li><br />
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<div style="display:none;" id="thursday"><br />
<br />
<br />
<!------------- Thursday -------------><br />
<h4> Ligation Efforts Continue</h4><br />
<ul><br />
<br />
<li>In addition to trying the triple-ligation strategy started on 7/7 (ligation attemp #3) will try trouble-shooting the ligation strategy used in attempts #1 and #2. In particular, will modify digestion protocol and test EcoRI and XbaI (older enzymes) for activity. </li><br />
</ul><br />
<b>Ligation Attempt #3 (Triple Ligation)</b> <br/><br />
<ul><br />
<li>Ran triple ligation all day at room temperature and stored at 4°C. Ligation contents were as follow to have a 1:1:1 stoichiometric ratio of all three components.</li><br />
<table><br />
<tr> <td><br />
Component </td> <td> Volume<br />
</td> </tr><br />
<tr> <td><br />
Distilled Water </td> <td>6.4 μL<br />
</td> </tr><br />
<tr> <td><br />
T4 Ligase buffer 10x </td> <td>2 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB T4 Ligase</td> <td> 1 μL<br />
</td> </tr><br />
<tr> <td><br />
pSB1C3 solution </td> <td> 2 μL<br />
</td> </tr><br />
<tr> <td><br />
B0015 solution</td> <td> 2.4 μL<br />
</td> </tr><br />
<tr> <td><br />
phsABC solution </td> <td> 6.2 μL<br />
</td> </tr><br />
<tr> <td><br />
Total </td> <td> 20 μL<br />
</td> </tr><br />
</table><br />
</ul><br />
<b>Ligation Attempt #4</b><br/><br />
<ul><br />
<li>Concerns about enzyme activity led to reversing the order of the serial digestion of B0015 background so that the first digestion is XbaI (low salt buffer) and the second is EcoRI (high salt buffer). Will also omit the pre-ligation PCR purification step.</li><br />
<li>Overnight digestion of ligation components--Set up the following digestions to run overnight at 37°C. The EcoRI was newly purchased as there was concern about the age of the previously used enzyme.</li><br />
<br />
<table><br />
<tr> <td><br />
<b>phsABC double digestion</b><br />
</td></tr><br />
<tr> <td> <br />
NEB EcoRI Buffer</td> <td> 5 μL<br />
</td></tr><br />
<tr> <td><br />
BSA 100x </td> <td>0.5 μL<br />
</td></tr><br />
<tr> <td><br />
phsABC solution </td> <td> 30.8 μL (1 μg DNA)<br />
</td></tr><br />
<tr> <td><br />
NEB EcoRI </td> <td>1.8 μL<br />
</td></tr><br />
<tr> <td><br />
NEB SpeI </td> <td>1.8 μL<br />
</td></tr><br />
<tr> <td><br />
Sterile H2O </td> <td> 10.1 μL<br />
</td></tr> </table><br />
<table> <tr> <td><br />
<b>B0015 digestion </b> <br />
</td></tr><br />
<tr> <td> <br />
NEB Buffer 4 </td> <td> 5 μL<br />
</td></tr><br />
<tr> <td><br />
BSA 100x </td> <td> 0.5 μL<br />
</td></tr><br />
<tr> <td><br />
NEB XbaI </td> <td> 3.6 μL<br />
</td></tr><br />
<tr> <td><br />
B0015 solution </td> <td> 3.8 μL ( 1 μg DNA)<br />
</td></tr><br />
<tr> <td><br />
Sterile H2O </td> <td>37.1 μL<br />
</td></tr><br />
</table><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 52 and 53 of the hard copy lab notebook.</i> <br />
<!------------- Thursday -------------><br />
</div><br />
<li><br />
<br />
Friday 7/9--Ligation attempt #4 to join phsABC and B0015 terminator.<br />
</li><br />
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<!------------- Friday -------------><br />
<h4> Ligation Attempt #4</h4><br />
<ul><br />
<li>Removed both overnight digestions, the XbaI digestion of B0015 and the EcoRI and SpeI digestion of phsABC, from 7/8 from the incubator.</li><br />
<li>Heatkilled enzymes of phsABC digest with 20 minutes at 80°C.</li><br />
<li>Ran <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/PCR_purify">PCR purification protocol </a>on XbaI digestion using microcentrifuge. In order to give a concentrated sample, eluted in only 35 μL of EB Buffer that had been heated at 50°C. Resulting concentration could not be determined as there was residual ethanol contamination.</li><br />
<li>Ran EcoRI digestion of purified XbaI digest product at 37 °C, with components as follows. Digestion was run for an hour before addition of CIP, after which it was run for another hour. </li><br />
<table> <tr> <td><br />
<b>B0015 digestion with EcoRI</b><br />
</td> </tr><br />
<tr> <td> <br />
NEB EcoRI buffer </td><td> 5 μL<br />
</td> </tr><br />
<tr> <td><br />
BSA 100x </td><td> 0.5 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB EcoRI </td><td>3.6 μL<br />
</td> </tr><br />
<tr> <td><br />
B0015 digested with XbaI </td><td>25 μL<br />
</td> </tr><br />
<tr> <td><br />
NEB Calf Intestinal Phosphatase </td><td>1 μL<br />
</td> </tr><br />
<tr> <td><br />
Sterile H2O </td><td> 15.9 μL<br />
</td> </tr><br />
</table><br />
<br />
Followed with 20 minute heatkill at 80°C.<br/><br />
<br />
<li>In order to set up ligation, need concentration of B0015, but ethanol contaminant continues to preclude measurement. Will estimate that the purification between digestion steps resulted in a 50% loss of DNA, leaving behind a 10 ng/μL concentration. Will also, as usual, run different ligation trials with different ratios of insert to vector, which should help combat any issues if this estimate is inaccurate </li><br />
<li>Set up the following ligation trials, with stoichiometric ratios of insert to vector (I:V) based on B0015 concentration estimate. Ran the ligations for 20 minutes at room temperature. </li><br />
<table><br />
<tr><td><br />
<b>Ligation Reaction Components</b><br />
</td></tr><br />
<tr><td><br />
Control Ligation</td> <td> 1:1 I:V </td> <td>2:1 I:V </td> <td> 3:1 I:V</td> <td> 4:1 I:V <br />
</td></tr><br />
<tr><td><br />
2 μL NEB T4 ligase buffer for all reactions<br />
</td></tr><br />
<tr><td><br />
1 μL NEB T4 ligase for all reactions<br />
</td></tr><br />
<tr><td><br />
5 μL B0015 solution for all reactions<br />
</td></tr><br />
<tr><td><br />
12 μL H<sub>2</sub>O </td> <td> 8.9 μL </td> <td>5.8 μL H<sub>2</sub>O </td> <td>2.7 μL H<sub>2</sub>O </td> <td> --<br />
</td></tr><br />
<tr><td><br />
-- </td> <td>3.1 μL phsABC </td> <td> 6.2μL phsABC </td> <td> 9.3 μL phsABC</td> <td> 12 μL phsABC<br />
</td> </tr><br />
</table><br />
<li> Following ligation, transformed 1 μL of each ligation reaction into commercial grade cells according to standard transformation protocol. Also transformed triple ligation reaction. </li><br />
</ul><br />
<br />
<h4>Enzyme Activity Test</h4><br />
<br />
To test enzyme activity, will do single digests of B0015 with enzymes in question and then run them on a gel to see if the plasmid linearized. XbaI and EcoRI are old, so will test them first. For XbaI activity test, simply set aside 10 μL of digestion solution from overnight digestion. Testing of EcoRI required additional digests. Digests using both old and new EcoRI samples were run for two hours at 37 °C with the following components:<br/><br />
<table><br />
<tr><td><br />
<b>EcoRI Activity </b><br />
</td> </tr><br />
<tr><td><br />
NEB EcoRI buffer </td> <td> 5 μL<br />
</td> </tr><br />
<tr><td><br />
BSA 100x </td> <td>0.5 μL<br />
</td> </tr><br />
<tr><td><br />
NEB EcoRI </td> <td>3.6 μL<br />
</td> </tr><br />
<tr><td><br />
undigested B0015 </td> <td>3.8 μL ( 1 μg DNA)<br />
</td> </tr><br />
<tr><td><br />
Sterile H<sub>2</sub>O </td> <td>37.1 μL<br />
</td> </tr><br />
</table><br />
<br />
Followed with 20 minute heatkill at 80°C.<br/><br />
<ul><br />
<li>Loaded all three enzyme activity tests on a 1.0% agarose gel and ran them at 90 V versus a 1 kb ladder and circular B0015 plasmid. </li><br />
<br />
Gel of single digests of B0015 Left to Right lane 1:1 kb ladder, lane 2: uncut circular B0015, its dimer, and lane 3 spillover, lane 3: XbaI digest of B0015, lane 4: old EcoRI digest of B0015, lane 5: new EcoRI digest of B0015<br/><br />
<br />
<li>Based on the results, all enzymes appeared to work, as they successfully created a linearized 3.2 kb fragment.</li><br />
<i>Wetlab work for this day is also recorded on pages 54-57 of the hard copy lab notebook. </i><br />
</ul><br />
<br />
<!------------- Friday -------------><br />
</div><br />
<br />
<br />
<li><br />
<br />
Saturday 7/10--Transformants from ligation attempts<br />
<br />
</li><br />
<br />
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<!------------- Saturday -------------><br />
<b> Transformants from ligation attempts #3 an #4</b> <br/><br />
<ul><br />
<li> The plate from ligation attempt #3 shoed in excess of 100 differnt colonies. </li><br />
<li> Plates from ligation attempt #4 had the following colony counts:</li><br />
Control-- 5 colonies <br/><br />
1:1--28 colonies <br/><br />
2:1--38 colonies <br/><br />
3:1--18 colonies <br/><br />
4:1--27 colonies <br/><br />
<li>Took colonies from various plates and used them to establish index plates on Amp LB, labeled as follow: eight colonies from triple ligation as #1-#8, four colonies from 1:1 as#9-#12, four colonies from 2:1 as #13-#16, four colonies from 3:1 as #17-#20, and four colonies from 4:1 as #21-#24.</li><br />
<i>Wetlab work for this day is also recorded on page 58 of the hard copy lab notebook.</i><br />
<!------------- Saturday -------------><br />
</div><br />
<br />
<li><br />
<br />
Sunday 7/11--Prep for copper growth assays of transformants<br />
</li><br />
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<!------------- Sunday -------------><br />
<b>Ligation work </b> <br/><br />
<ul><br />
<li> Realize that mistakenly plated triple ligation reaction on ampicillin plate rather than the required chloramphenicol, so none of the colonies present are of value. Fortunately still have transformation solution, so replate it, this time on a chloramphenicol </li><br />
<li> Inoculated overnight liquid cultures of ligation attempt #4 transformants (#9-#24 on index plates) for miniprep the following morning </li><br />
<b>Growth assay prep work </b> <br/><br />
<li> Want to see if pSB74 confers any copper resistance on <i>E. coli</i>, since it should help them precipitate nearby copper and thus reduce the copper concentration that they experience. </li><br />
<li> Inoculated liquid cultures of LE392 with pSB74 in ampicillin LB and untransformed LE392 in plain LB and left to grow overnight on shaker at 37˚C for growth assays the following day </li><br />
<li> Prepared CuSO<sub>4</sub> solutions in LB. Each well in the 96-well plate will have 225 uL of copper solution and 25 uL of cell solution, so the copper solution must have a CuSO<sub>4</sub> concentration that is 10/9 the desired final values. The copper solutions must also contain ampicillin and IPTG at the same levels as the cultures that will be introduced. The desired CuSO<sub>4</sub> concentrations are those used in the wide and narrow concentration range trials on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> 6/10 </a> and <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_2"> 6/14 </a>. </li><br />
</ul><br />
<i>Wetlab work for this day is also recorded on pages 59-61 of the hard copy lab notebook.</i><br />
<!------------- Sunday -------------><br />
</div><br />
<br />
<!------------- LAB NOTEBOOK -------------><br />
</ul><br />
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