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http://2010.igem.org/Team:Gothenburg-Sweden/Results
Team:Gothenburg-Sweden/Results
2010-10-03T12:03:55Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
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<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<br />
<p>coming soon...</p><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> <br> <br />
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<div class="Title3">Lab Notes</div><br />
<br />
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<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
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<br />
<br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br></p><br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
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<br />
<br />
<br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note
Team:Gothenburg-Sweden/Lab Note
2010-10-03T12:03:34Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
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<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/f/fe/Gfp.png" width="40" height="40" align="left"></div> <strong><p>Fluorescent Proteins:</strong><br> Green Fluorescent Protein <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p><br> <br />
<br />
<div class="logo"> <img src=" https://static.igem.org/mediawiki/2010/a/a9/Plasmid.png" width="40" height="40" align="left"></div><p><strong>Plasmid backbone:</strong> <br><br />
pSP-GM1 <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#plasmid">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/7/74/Snf1Com.png" width="40" height="40" align="left"></div><p> <strong>SNF1 (modified subunits):</strong><br><br />
Snf1 (alfa), Snf4 (gamma) <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#SNF1">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/4/46/FpPos.png" width="40" height="40" align="left"></div> <p> <strong>FP positions:</strong> <br><br />
N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#position">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/e/e0/PrDesign.png" width="40" height="40" align="left"></div> <p> <strong>Primer design:</strong><br> <br />
Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#primer">(read more)</a></p> <br> <br />
<br />
<br />
<br />
<div id="myTitle">Lab work by date </div> <br />
<ul><li><p> <strong>2010-08-11</strong><br> <br />
Malin and Katarina<br> <br />
<br> <br />
The PCR reaction from yesterday was checked on a gel. α should be around 1500 bp but only a band around 750 bp was seen. We had a long meeting with supervisor Per about PCR reactions and the next couple of weeks in the lab. <br> <br />
<br> <br />
Karl and Kemal <br> <br />
<br> <br />
Ran PCR reaction α with different settings. <br> <br />
<strong>α1: </strong>regular settings<br> <br />
<strong>α2: </strong>10 x diluted template 2<br> <br />
<strong>α3: </strong>increased annealing time from 30 s to 60 s<br> <br />
<strong>α4: </strong>decreased annealing temperature<br> <br />
<br> <br />
After PCR, the products were run on a gel. α1, α3 and α 4 had a band around 750 bp but nothing on α2. </p></li><br> <br />
<br> <br />
<li><p> <strong>2010-08-10</strong><br> <br />
Peidi and Lokesh<br> <br />
<br> <br />
Checked the transformation plates but nothing had grown. Prepared PCR reaction Γ. <br> <br />
<br> <br />
Katarina and Kemal<br> <br />
<br> <br />
PCR product Γ was verified on a 0.7 % agarose gel. There was a band around 750 bp as expected and it was purified using the regular protocol. A new phusion mastermix was made and PCR reaction α was run with Γ as one of the template. Regular phusion PCR settings was used. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-08-09</strong><br> <br />
Malin and Kemal<br> <br />
<br> <br />
Before checking if the ligation worked we need to transform and amplify in E.coli. Then we can miniprep the ligated plasmid and check on a gel. After that we can digest plasmid with PacI and BcuI and ligate the digested IV. Then another round of transformation and miniprep before we can transform yeast. <br> <br />
<br> <br />
Karl and Lokesh<br> <br />
<br> <br />
Transformed competent E.coli with plasmid ligated with III by heat shock. The cells were incubated at 37 °C over night. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-07</strong><br> <br />
Lokesh<br> <br />
<br> <br />
Moved ligation tube to freezer. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-06</strong><br> <br />
Malin, Julia and Lokesh<br> <br />
<br> <br />
III and IV were digested 3x protocol and were then put on a 1 % agarose gel together with Γ both for verification and purification. Correct bands were seen for purification except for Γ which was empty. In the verification lanes the digested PCR products were to light to be seen but they were seen in purification lanes. IIId and IVd were purified. <br> <br />
<br> <br />
<strong>IIId: </strong>7.8 ng/µl<br> <br />
<strong>IVd: </strong>11.7ng/µl<br> <br />
<br> <br />
IIId will now be ligated with the cut plasmid (BamHI and HindIII). Ligation performed according to protocol with 156 ng insert and 52 ng plasmid. Ligation was incubated over night in fridge at 4-16° C. <br> <br />
<br> <br />
All 4 sporulation plates were checked but still no tetrads visible. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-05</strong><br> <br />
Malin and Julia<br> <br />
<br> <br />
III and IV were digested using the regular protocol. <br> <br />
<strong>III: </strong>HindIII and BamHI works with Buffer B<br> <br />
<strong>IV: </strong>PacI and BcuI works with Buffer M<br> <br />
<br> <br />
A 1% gel was run with uncut and digested III and IV, control, I and Β. I worked well and will be purified. The Β lane showed no bands. None of the digested products were visible on the gel so a new gel was run trying to verify this result. The control shows a band around 1700 which is weird. This band is also visible on the III uncut but together with a band around 750 bp. No bands at the IV which is very strange since this PCR product has been verified before. Have something gone wrong with the purification? <br> <br />
<br> <br />
As a result PCR reactions III and IV will be redone. New PCR reactions of Γ and α were also performed in the same block. <br> <br />
<br> <br />
Karl and Kemal<br> <br />
<br> <br />
Reactions of Β were made. Β1 is a continuation of the 100 cycle PCR this time with primers. Β2 uses a megaprimer approach. <br> <br />
<br> <br />
All the PCR products were run on a 1 % agarose gel. III, IV and Γ were alright. α and Β2 showed a band around 750 bp. <br> <br />
<strong>III: </strong>531.3 ng/µl<br> <br />
<strong>IV: </strong>263.9 ng/µl<br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-04</strong><br> <br />
Julia and Karl<br> <br />
<br> <br />
PCR products Β were loaded on a 0.7 % gel. No correct bands, only amplified templates around 750 bp and something else around 500 bp. <br> <br />
<br> <br />
PCR of SAMS will also be redone from scratch. PCR reactions 1, 2 and C are done, then loaded on a gel (2 % gel and low range ladder) and purified. <br> <br />
<strong>1: </strong>8.2 ng/µl<br> <br />
<strong>2: </strong>7.8 ng/µl<br> <br />
<strong>C: </strong>4.9 ng/µl<br> <br />
Samples diluted to 1 ng/µl. <br> <br />
<br> <br />
PCR of Β was done again but this time in two steps and with the Finnzymes Tm which corresponds to an annealing temp at 85 °C. Firstly 10 cycles without primers were performed. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
Second round of PCR with primers were performed. Then new PCR reactions amplifying III and IV were done and also reaction I. <br> <br />
<strong>III: </strong>572 ng/µl<br> <br />
<strong>IV: </strong>660 ng/µl<br> <br />
<br> <br />
The sporplates were checked and very few tetrads were seen. <br> <br />
<br> <br />
Β samples were put on a gel but no correct bands visible. We suspect that the reverse primer is attaching in the wrong place since YFP and CFP is so similar which then would lead to 750 bp fragment. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-03</strong><br> <br />
Malin and Lokesh<br> <br />
<br> <br />
PCR products II, III and IV were verified and purified from a 1 % agarose gel. III did not work and was not purified. <br> <br />
<br> <br />
Concentrations: <br> <br />
<strong>II: </strong>6.2 ng/µl <br> <br />
<strong>IV: </strong>5.6 ng/µl<br> <br />
II and IV were diluted to 1 ng/µl. <br> <br />
<br> <br />
The new sporplates were examined but no tetrads visible. To check further the cells were stained with DAPI and examined in fluorescence microscope. Still no success. <br> <br />
<br> <br />
New PCR reactions were prepared. III was redone and Β was also done. Same settings as yesterday. <br> <br />
<br> <br />
Kemal and Karl<br> <br />
<br> <br />
PCR products were loaded on a 1% agarose gel for verification and purification. III worked this time and was purified from the gel using the protocol from QIAquick Gel Extraction Kit. <br> <br />
<strong>III: </strong>6.4 ng/µl<br> <br />
<br> <br />
New PCR of Β was run with two different block settings. <br> <br />
<strong>Block A: </strong>increased annealing time to 1 min. <br> <br />
<strong>Block B: </strong>increased annealing time to 58 °C. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-02</strong><br> <br />
Kemal, Lokesh and Karl<br> <br />
<br> <br />
PCR products A, B and 3 were loaded on a gel and then purified from the gel using the protocol from QIAquick Gel Extraction Kit. α and IV were also run on a gel but only showed strong bands corresponding to amplified templates. On the α lane however a very light band was seen at the correct length. This will be further investigated. <br> <br />
<br> <br />
Malin and Julia<br> <br />
<br> <br />
The purified PCR products were measured with nanodrop: <br> <br />
<strong>A1: </strong>11.8 ng/µl<br> <br />
<strong>A2: </strong>10.2 ng/µl<br> <br />
<strong>B1: </strong>9.6 ng/µl<br> <br />
<strong>B2: </strong>17.1 ng/µl<br> <br />
<strong>31: </strong>15.0 ng/µl<br> <br />
<strong>32: </strong>16.3 ng/µl<br> <br />
<br> <br />
A, B and 3 were diluted to 1 ng/µl. <br> <br />
<br> <br />
PCR products 4 and 5 were loaded on a 1 % agarose gel together with IV that should be controlled. PCR products 4 and 5 were correct so they were purified from the gel using the protocol from QIAquick Gel Extraction Kit. IV however was not correct. Only a band around 750 bp was visible. <br> <br />
<br> <br />
Concentrations: <br> <br />
<strong>4: </strong>10.8 ng/µl<br> <br />
<strong>5: </strong>10.4 ng/µl<br> <br />
4-5 were diluted to 1 ng/µl. <br> <br />
New PCR reactions were started (II, III and IV). PCR settings that works well with the Phusion enzyme was set: <br> <br />
Initial denaturation: 98 °C 3 min<br> <br />
Denaturation: 98 °C 10 s<br> <br />
Annealing: 55 °C 30 s<br> <br />
Elongation: 72 °C 1.5 min+ 1s/cycle<br> <br />
30 cycles<br> <br />
Final extension: 72 °C 10 min<br> <br />
4°C forever <br> <br />
<br> <br />
<strong>2010-07-30</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
The concentration of the purified plasmids were measured with nanodrop: <br> <br />
<strong>P1: </strong>1.7 ng/µl<br> <br />
<strong>P2: </strong>3.5 ng/µl<br> <br />
<strong>P3: </strong>25.9 ng/µl<br> <br />
<strong>P4: </strong>42.8 ng/µl<br> <br />
This is clear evidence that water should be used for eluation in the future! <br> <br />
<br> <br />
To make sure that we have the right products after purification a 1 % agarose gel was run with fastruler DNA ladder. The gel verified the plasmid size though the bands were light. <br> <br />
<br> <br />
PCR product III was evaporated since it was so dilute. New concentration: <br> <br />
<strong>IIId: </strong>35 ng/µL<br> <br />
<br> <br />
The digested PCR product III and the digested plasmid were ligated with T4 ligase. The DNA concentration in total should not exceed 1 µg. The ratio insert:vector should be 3:1. <br> <br />
<br> <br />
<strong>Ligation protocol: </strong><br> <br />
150 ng insert<br> <br />
50 ng vector<br> <br />
3 µl 10x T4 buffer<br> <br />
2.5 µl (2.5 U, between 1-5 U) T4 ligase<br> <br />
Adjust volume with water up to 30 µl. Mix and incubate in 4-16 °C (refrigerator) overnight. <br> <br />
The newly plated sporplates were moved to 30°C. The old sporplates were again studied in the microscope and no tetrads were seen. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
Since we have had a lot of trouble with the longer fusions we will start over from scratch with a new high fidelity polymerase called “Finnzumes Phusion”. New PCR reactions were run (A, B, 3, 4 and 5). IV and α were also performed using alternative settings (2 different PCR reactions described before) but with “old” templates. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-29</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
PCR product III is the final fusion protein where Snf4 is tagged with yfp. To be able to insert the construct into the plasmid we must digest it. <br> <br />
<br> <br />
Digestion: Mix DNA, buffer and water. Last add enzyme and mix. Incubate at 37° C for 1 hour. <br> <br />
1 µg DNA<br> <br />
2.5 µl Buffer<br> <br />
1 U Enzyme<br> <br />
Up to 25 µl H2O<br> <br />
For PCR product III, the restriction enzymes BamHI and HindIII are used together with buffer B. After digestion (the protocol was scaled 3x) the sample was loaded on a 1 % agarose gel in 6 lanes with 1 kb ladder. The bands were cut out and weighed. The samples were purified from the gel using the protocol from QIAquick Gel Extraction Kit. The DNA was eluated with water. <br> <br />
<br> <br />
Concentration: <br> <br />
<strong>IIId: </strong>5 ng/µl (Total product 160 µl= 800 ng) <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
The plasmids were also digested with BamHI and HindIII according to the same protocol as above. P1 and P2 were eluated with buffer EB and P3 and P4 with water. <br> <br />
<br> <br />
2 new sporulation plates were prepared by plating the diploid yeast cells on spore plates and kept at room temperature. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-28</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
The PCR products were run on a 1 % gel with 1 kb ladder. Γ may have worked. α and Β had a band around 750 bp and a weird light band around 500 bp. They obviously did not work. IV did not work at all. <br> <br />
<br> <br />
The sporulation plates were checked for spores. Cells were studied under a microscope. No tetrads visible. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-07-27</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
A 1.5 % gel was run with all of the fragments around 700 bp to control the validity of the PCR reactions. A low range ladder was used. PCR products 1-5 seems good. The results from yesterday were confirmed. <br> <br />
<br> <br />
A new PCR design was made with α, Β, Γ and IV. PCR was done in two steps to increase the chance of success for longer fusions. <br> <br />
1. 7 cycles with short annealing time. <br> <br />
2. 20 cycles with long annealing time (all other settings kept standard). <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-26</strong><br> <br />
<br> <br />
A new experiment were designed called Γ which is the same as I but with longer annealing region. Also an experiment called Δ were designed which corresponds to II. <br> <br />
<br> <br />
Julia and Katarina<br> <br />
<br> <br />
PCR reactions were run with samples of 4, IV and Γ. IV was tried with two different PCR settings: one regular and one with two runs of PCR, the first without primers and only 5 cycles and the second one with regular settings. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
The PCR products were controlled on a 0.7% agarose gel. Only 4 worked. Γ was empty and IV had bands in the wrong place (amplified template). <br> <br />
<strong>4: </strong>534 ng/ µl </p></li> <br />
<li><p> <strong>2010-07-22</strong><br> <br />
Karl, Lokesh and Kemal<br />
</br></br> <br />
A new mastermix was prepared using a different enzyme. Then the experiments from yesterday was redone using regular PCR settings: <br> <br />
A, 5, 2, I<br> <br />
Then some more experiments of IV was redone: <br> <br />
2a, 2b and 2c<br> <br />
The new primer 14 had arrived so PCR reaction 4 was also redone. <br> <br />
<br> <br />
All the PCR products were run on a 0.7 % agarose gel with GeneRuler 1 kb DNA ladder. The results were inconclusive. None of the expected bands were received. At this point we are not sure what to do since none of the PCR reactions worked. <br> <br />
<br> <br />
</p> <br />
</li> <br />
<li> <p> <strong>2010-07-21</strong><br> <br />
Malin, Julia, Karl and Lokesh<br> <br />
<br> <br />
<strong>Verification of the PCR products </strong>α1, α2, α3, α4, α5 and αK by running on a thin 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder). <br> <br />
<br> <br />
The gel didn’t show the expected band lengths. A band around 700 bp could be seen, most strongly for α4 and α5. We suspect that there is something wrong with the ladder because we get inconclusive results. Since none of the PCR reactions for the last couple of days have worked we will redo the templates of α and IV: I, 2, 5 and A. These PCR reactions have worked before. <br> <br />
<br> <br />
<strong>Expected fragment length: </strong><br> <br />
A.2: 1938 bp<br> <br />
5.2: 757 bp<br> <br />
2.2: 762 bp<br> <br />
I.2: 768 bp<br> <br />
<br> <br />
The PCR products were run on a thin 0.7 % agarose gel. We used both the FastRuler High Range Ladder and the GeneRuler 1 kb DNA ladder. 5.2 and 2.2 showed the expected bands around 750 bp. I.2 and A.2 did not work at all, nothing visible on the gel. We also run some of the old PCR products to see what the result would be using a new ladder. We run α2, α5, 4c (IV) and 4c*(IV). Here some template amplifications were visible but sadly none of the expected fragment lengths. Both ladders showed the same result so there is probably nothing wrong with the GeneRuler ladder except that it is not showing strong color. <br></p></li> <br />
<li><p><strong>2010-07-20</strong><br> <br />
Malin, Julia, Lokesh and Karl<br> <br />
<br> <br />
<strong>Verification of yesterdays PCR reactions </strong>by running on a 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder) <br> <br />
<br> <br />
None of the reactions worked. <br> <br />
<br> <br />
Gel 1 (Block A): Nothing visible on gel, ladders only very light so these samples were run on a new 0.7 % agarose gel but this time thinner to get better resulotion (0.58 g agarose + 80 ml TBE). This time no expected fragments were seen, but bands corresponding to primer dimers were clearly seen. <br> <br />
Gel 2 (Block B): A few small bands were seen but no bands of expected length. <br> <br />
<br> <br />
We decided to stop working on IV until proper feedback on the experimental setup was received. Instead we continued with α.<br> <br />
<br> <br />
<strong>Doing another PCR round of α (SAMS peptide): </strong> <br> <br />
<strong>α: PCR fusion of cfp-SAMS and yfp: </strong>Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br> <br />
<br> <br />
α1: regular protocol. <br> <br />
α2: regular protocol. <br> <br />
α3: 1.25 µl extra primer 9.<br> <br />
α4: 1.25 µl extra primer 12. <br> <br />
α5: 1.25 µl extra primer 9 and 1.25 µl primer 12. <br> <br />
αK: Control<br> <br />
Regular PCR-settings</p></li> <br />
<li><p> <strong>2010-07-19</strong><br> <br />
Karl, Peidi and Kemal<br> <br />
<br> <br />
<strong>Doing a new experimental setup for IV: </strong><br> <br />
<strong>IV. PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br> <br />
<br> <br />
13 different tubes were prepared and run at two different PCR-settings. Block A: Regular settings except double annealing time (1 min). Block B: Regular settings except 58° C annealing temperature. <br> <br />
<br> <br />
C : Control<br> <br />
1a: same as 2* (template 5 5 x diluted) <br> <br />
1b. same as 3* (template 5 10 x diluted) <br> <br />
1c: same as 4* (template 5 100x diluted) <br> <br />
2a: increased product A concentration to 5 ng/µl. <br> <br />
2b: increased product A concentration to 10 ng/µl. <br> <br />
3a: same as 5* (2x primer 7) <br> <br />
3b: same as 6* (template 5 diluted 5x, 2x primer 7) <br> <br />
3c: same as 7* (template 5 diluted 10x, 2x primer 7) <br> <br />
3d same as 8* (template 5 diluted 100x, 2x primer 7) <br> <br />
4a: 2x primer 7. <br> <br />
4b: 2x primer 7, 5x template A. <br> <br />
4c: 2x primer 7, 10 x template A. <br></p></li> <br />
<li><p> <strong>2010-07-16</strong><br> <br />
Lokesh, Kemal, Katarina and Peidi<br> <br />
<br> <br />
<strong>Verification of PCR </strong>reactions IV* (8 tubes) and α on a 0.7 % agarose gel (5 µl sample+ 2µl loading buffer, GeneRuler 1 kb DNA ladder). <br> <br />
<br> <br />
No bands visible. None of the PCR reactions worked, just small bands visible corresponding to primers. The α PCR reaction messed the tubes up. That might be an explanation why there were no bands on the gel for α. A new experimental setup must be made tomorrow. <br> <br />
<br> <br />
PCR products I and 2 were run with a low range ladder to see that those products are ok. That gel was satisfactory. <br> <br />
<br></p> <br />
<li><p><strong>2010-07-15</strong><br> <br />
Malin, Julia, Kemal and Karl<br> <br />
<br> <br />
<strong>Concentrations of PCR products: </strong><br> <br />
4.2. 356.2 ng/µl<br> <br />
I. 338.3 ng/µl<br> <br />
II. 303.6 ng/µl<br> <br />
III. 337.5 ng/µl<br> <br />
IV. 280.9 ng/µl<br> <br />
<br> <br />
All PCR-products were diluted to 1 ng/µl. <br> <br />
<br> <br />
<strong>Verified PCR products</strong> I-IV and 4.2 on a 0.7 % Agarose Gel (8 µl sample + 2µl loading buffer, GeneRuler 1 kb DNA ladder) <br> <br />
PCR products I, II and III were good but 4.2 showed no bands at all. After primer design control we found a problem with primer 14, which then was ordered again. IV had only a band around 700 bp instead of 2600. This might be template 5, which then has been amplified. If the settings are changed maybe it will work. Firstly the template 5 will be diluted 0x, 5x, 10x and 100x (4 tubes: 1*, 2*, 3* and 4*) and these dilutions will also be run with higher primer concentration 7 (4 tubes: 5*, 6*, 7* and 8*). 2.5 µl primer 7 will be added instead of 1.25 µl primer 7. <br> <br />
<br> <br />
New PCR reactions were run: <br> <br />
<strong>α: PCR fusion of cfp-SAMS and yfp: </strong> Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br> <br />
K4. Control. <br> <br />
<br> <br />
<strong>IV *: PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br> <br />
Half volume 25 µl. Regular PCR settings except 56 °C annealing temperature. <br> <br />
1*;- 8*<br> <br />
K1*. Control for 1-4*.<br> <br />
K2*. Control for 5-8*.<br> <br />
<br></p></li> <br />
<br />
<li> <p><strong>2010-07-14</strong><br> <br />
Julia and Katarina <br> <br />
<strong>Ran PCR 1-5.</strong><br> <br />
1. Amplification of cfp-sams compatible: Add restriction enzyme site BamHI on N-terminus +SAMS on C-terminus.<br> <br />
Expected fragment length: 748 bp.<br> <br />
2. Amplification of yfp-SAMS competible: Adds restriction enzyme site for HindIII on C-terminus and SAMS tail on N-terminus. <br> <br />
Expected fragment length: 762 bp.<br> <br />
3. Amplification of yfp-snf4 compatible: Adds restriction enzyme site BamHI on N-terminus and Snf4 tail on C-terminus. <br> <br />
Expected fragment length: 755 bp.<br> <br />
4. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. <br> <br />
Expected fragment length: 769 bp.<br> <br />
5. Amplification of cfp-snf1 compatible: Adds restriction enzyme site SpeI on N-terminus and Snf1 tail on C-terminus.<br> <br />
Expected fragment length: 757 bp.<br> <br />
K2. Control K2.<br> <br />
<br> <br />
Julia, Katarina and Malin<br> <br />
<strong>Verified PCR 1-5:</strong> measured concentration by nanodrop and checked if the fragments were the correct length by running on a 2 % agarose gel (8 µl sample+2 µl loading buffer, Fermentas FastRuler Low Range).<br> <br />
PCR product 1, 2, 3 and 5 worked and gave expected fragment lengths. Number 4 however did not work. Only a fragment around 80 bp was visible, probably a primer dimer.<br> <br />
<br> <br />
<strong>Concentrations:</strong><br> <br />
1. 443.9 ng/µl<br> <br />
2. 371.0 ng/µl<br> <br />
3. 361.7 ng/µl<br> <br />
4. --<br> <br />
5. 377.6 ng/µl<br> <br />
A. 451.1 ng/µl (snf1)<br> <br />
B. 438.7 ng/µl (snf4)<br> <br />
C. 408.9 ng/µl (SAMS) <br> <br />
1-5 and A-C were diluted to 1 ng/µl.<br> <br />
<br> <br />
<strong>Ran PCR of I, II, III, IV and 4.</strong><br> <br />
4.2. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. Expected fragment length: 769 bp. (second try)<br> <br />
I. PCR fusion of cfp and SAMS: Adds BamHI on N-terminus of cfp and gives a SAMS C-terminus. Expected fragment length: 768 bp.<br> <br />
II. PCR fusion of yfp and Snf4 1: Adds BamHI on N-terminus of yfp and removes stop codon. Expected fragment length: 1694 bp.<br> <br />
III. PCR fusion of yfp and Snf4 2: Adds BamHI on N-terminus of yfp and adds HindIII on C-terminus of Snf4 plus adds stop codon. Expected fragment length: 1708 bp. Final Product!<br> <br />
IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.<br> <br />
K3. Control<br> <br />
<br> <br />
Karl and Malin<br> <br />
Transferred diploid cells of snf1Δ mutants to two sporulation plates and incubated in 30° C. (For 2-7 days)<br> <br />
</p></li> <br />
<br />
<li><p><strong>2010-07-13</strong><br> <br />
Julia and Lokesh<br> <br />
Ran the gel with products of our first three PCRs along with the templates we made for the two fluorescent proteins. We also prepared the sample solutions of DNA templates with appropriate concentration (1ng/µl) for the next PCR reactions.<br> <br />
<strong>Verified</strong> the PCR products and the digested miniprepped EYFP and ECFP by running an agarose gel which showed good results. Expected fragment lengths were received. The SAMS-peptide however was too short to be visible on the gel (67 bp).<br> <br />
<strong>Prepared for PCR 1-5. </strong>The miniprepped ECFP and EYFP were diluted to a stock concentration of 1 ng/ ml to use for the PCR. The concentrations of the first PCR product were measured using nanodrop and then diluted to 1 ng/ml.<br> <br />
snf1: 20.5 ng/ml<br> <br />
snf4: 122.6 ng/ml<br> <br />
SAMS: 114.9 ng/ml<br> <br />
</p> <br />
<p> Karl and Adnan<br> <br />
We made duplicates of the haploid Snf4∆ and wildtype strains on new plates in case the original plate would be contaminated. Prepared a sporulation medium that was autoclaved and poured into empty growth plates to stay in room temperature over night.</p> <br />
<br></li> <br />
<li> <p> <strong>2010-07-12</strong><br> <br />
Katarina and Julia<br> <br />
<strong>Amplification of genomic SNF1 DNA by PCR. </strong>All primers were resuspended in mq-water according to the volume on the lable, the final concentration 100 pg/ml. The primers were then diluted 10x and the SAMS-peptide templates were diluted 1000x. PCR reactions were run amplifying subunits Snf1 and Snf4 and also the SAMS-peptide. YPD medium and agar plates were prepared (yeast).<br> <br />
</p> <br />
<p> Adnan and Kemal<br> <br />
<strong>Miniprep purification </strong>of EYFP and ECFP plasmides from overnight culture. The concentrations were measured using Nanodrop:<br> <br />
ECFP1: 99 ng/ml<br> <br />
ECFP2: 89.9<br> <br />
EYFP1: 104, 7 <br> <br />
EYFP2: 172,5<br> <br />
The concentrations were verified with electrophoresis. The plasmids were cut with restriction enzymes EcoRI and Pst1.<br> <br />
ECFP, plasmid pSB1AK3, fragment sizes 750 bp and 3200 bp.<br> <br />
EYFP, plasmid pSB1A2, fragment sizes 750 bp and 2050 bp.</p></li> <br />
<br> <br />
<li><p><strong>2010-07-11</strong><br> <br />
Katarina<br> <br />
Made overnight cultures with colony 3 and 4 plasmid (not single cultures) and twp each of EYFP and ECFP.<br />
</p> <br />
</li> <br />
<br> <br />
<li><p><strong>2010-07-10</strong><br> <br />
Julia<br> <br />
Moved plated bacteria from incubator to refrigerator. <br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-09</strong><br> <br />
Malin and Julia<br> <br />
The SOC medium was autoclaved. Competent E.coli cells were transformed with our FPs through heat shock and plated on petri dishes. 20 new LB-amp plates were made.<br> <br />
<br />
Adnan and Katarina<br> <br />
Picked up primers at Chalmers and checked if they were ok. The concentration of the genomic DNA (SNF1 gene) were measured. Prepared a master mix so that the PCR-reactions could be started Monday morning.</p> <br />
<br> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-08</strong><br> <br />
Malin, Julia and Peidi<br> <br />
The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-07 </strong><br> <br />
Adnan, Katarina and Julia<br> <br />
As the gels from Tuesday showed ambigous results we decided to redo the miniprep of the plamids. We used the overnight preparations of the two unused colonies (3 &amp; 4). The miniprep went smoothly except for major difficulties regarding untrustworthy pipettes. The pipettes marked with green tape are the ones to use from now on together with the tips with the same markings! A gel was molded and this time we decided to use a full gel to get a better resolution. The results on the gel were very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-06</strong><br> <br />
Malin, Adnan and Peidi<br> <br />
Colony 1 and 2 were picked to do a miniprep. The plasmids were extracted and a plasmid integrity check was performed. By cutting with BamHI, one cut were made to get the size of the plasmid. Then we cut with ClaI which is a two-cutter that gives a fragment that is 600 bp and one that is 6800. The gel which should confirm this was not satisfactory since the 600 band was missing.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-05</strong><br> <br />
Malin and Katarina<br> <br />
An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1.</p> <br />
</li> <br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Contact
Team:Gothenburg-Sweden/Contact
2010-10-03T11:58:09Z
<p>Sanli k: </p>
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<p>Please do not hesitate to contact us if you have any questions or suggestions regarding our project. <br />
We are available at: igem_goteborg_2010@googlegroups.com</p><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Sponsors
Team:Gothenburg-Sweden/Sponsors
2010-10-03T11:57:42Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
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<a href="#">Chalmers University of Technology</a><br />
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<br />
<p>We are currently looking for an official corporate sponsor that is interested in the field of synthetic biology and the major medical applications our technology could lead to. We have good experience of collaborating with the industry, and if you choose to partner up with us we can assure you that your company will have great international exposure during the iGEM contest. Please contact us at burmank@student.chalmers.se and we can discuss the terms of our companionship.</p><br />
<br><br><br><br><br><br><br><br><br><br><br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<br />
<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/About_us
Team:Gothenburg-Sweden/About us
2010-10-03T11:57:21Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a><br />
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<br />
<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<div id="text"><a name="intro"></a><br />
<p> We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines the cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing a reporter mechanism for cellular stress in yeast by tagging protein and peptides affiliated with the stress activated SNF1 complex with fluorescent markers. </p> <br />
<br />
<br />
<br />
<br />
<div id="myTitle">Chalmers University of Technology <a name="cth"></a> </div> <br />
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<br />
<p><br />
<strong>Chalmers in central Göteborg on the Swedish West Coast</strong></p> <br />
<br />
<p>Chalmers is a university of technology in which research and teaching are conducted on a broad front within technology, natural science and architecture. Our inspiration lies in the joy of discovery and the desire to learn. Underlying everything we do is a wish to contribute to sustainable development both in Sweden and world-wide.<br> <br />
<br> <br />
Our research work deals ultimately with improving people’s conditions, and we often cross traditional boundaries in order to solve the problems of the future. Chalmers has become strong within several areas of science, and some of our research leads its field internationally. We wish in particular to develop and strengthen our research in the fields of bioscience, information technology, environmental science and nanotechnology.<br> <br />
<br> <br />
Chalmers is a pioneer in Europe in the field of start-up companies, and has built up an efficient system of innovation in order to find applications in research into the formation of companies. <br> <br />
<br> <br />
Chalmers has great aspirations to develop continually by opening doors to the outside world and by thinking innovatively. We have a close and productive collaboration with business and society at large. In this way we reinforce our own development, at the same time as we contribute to the growth of society.<br> <br />
<br> <br />
Chalmers was founded in 1829 as a result of a donation from the director of the Swedish East India Company, William Chalmers. His motto, and ours, is:<br> <br />
<br> <br />
Avancez!</p> <br />
<br />
<br />
<br />
<br />
<div id="myTitle">Supervisors<a name="svs"></a></div> <br />
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<td width="136"><span class="style9">Marcus Wilhelmsson </span></td> <br />
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<td width="237">&nbsp;</td><br />
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<tr><br />
<td colspan="2" valign="top"><table width="363" height="150" border="1"><br />
<tr><br />
<td width="147"><span class="style4">Age:</span></td><br />
<td width="158"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">Exchange year at University of Minnesota, MS Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"> <br />
<tr> <br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/7/7f/Katarina.jpg" alt="" width="133" height="200" class="img1" /></span></td> <br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"> <br />
<tr> <br />
<td><span class="style9">Katarina Risö </span></td> <br />
</tr> <br />
<tr> <br />
<td><span class="style11">student</span></td> <br />
</tr> <br />
</table></td> <br />
<td width="237">&nbsp;</td> <br />
</tr> <br />
<tr> <br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Chemical Engineering</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Chemistry and Biosciences</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table><br />
</td> <br />
</tr> <br />
</table> <br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/a/a7/Peidi.jpg" alt="" width="133" height="200" class="img1" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td><span class="style9">Peidi Liu </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Chinese</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">Electronic Engineering</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics </span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/8/80/Adnan.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td><span class="style9">Adnan Kadic</span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Bosnian</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Chemistry and Biosciences </span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/ea/Lokesh.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="221" height="59" valign="top"><table width="216" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="180"><span class="style9">Lokeshwaran Manoharan </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="124">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">23</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Indian</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B.Tech Ind Biotechnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/0/01/Kemal.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Kemal Sanli </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Turkish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Molecular Biology and Genetics </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/4/40/Karl.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Karl Almén Burman </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">23</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotechnology </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Biotechnology</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/e8/Julia.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Julia Fransson </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">22</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotechnology </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">Exchange year through Unitech International</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<p>&nbsp;</p> <br />
</td><br />
</tr><br />
<tr><td>&nbsp;</td> <br />
</tr><tr><td>&nbsp;</td> <br />
</tr> <br />
</div><br />
<br />
</div> <br />
</div><br />
<div id="right"><br />
<div class="Title3">Lab Notes</div><br />
<br />
<br />
<div id="lab"><br />
<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
<br />
<br />
<br />
</div> <br />
<div class="Title3">News</div><br />
<div id="news"><br />
<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
</div> <br />
<br />
<br />
<br />
</div><br />
<br />
<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
<br />
</div><br />
</div></div> <br />
</body><br />
</html></div>
Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Project
Team:Gothenburg-Sweden/Project
2010-10-03T11:57:00Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#">Methods</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden">Home</a></li><br />
<li id="active"><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#aim">Aim</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#bkg">Theoretic background</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#method">Methods</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#tech">Techniques</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#snf1">SNF1 &amp; AMPK </a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#bricks">Biobricks</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#app">Possible Applications</a></li> <br />
</ul> <br><br />
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<br />
<div id="myTitle">Project Description </div> <br />
<br />
<p>The conceptual idea is to use the conformational change in the SNF1 protein complex to establish a FRET (Förster Resonance Energy Transfer) system for cellular stress detection. There are two chromophores tagged at appropriate locations and upon undergoing the conformational change, they come in close proximity such that the emission energy from one can excite the other, thus resulting in different emission energy. There are two possibilities to be tested: <br><br>1. To fuse the chromophores to the subunits of the Snf1 complex to build an active readout of the conformational change <br> <br />
2. To fuse the chromophores to a short peptide (SAMS peptide) that can be phosphorylated by Snf1 and is commonly used as a read-out of Snf1 activity. </p> <br> <br />
<br />
<br />
<br />
<ul id="myList"> <li><p> <strong>Working package 1: Find positions for fluorescent markers in SNF1 </strong><br> <br />
For WP1 all present information about the protein complex is utilized, so that the fluorescent tags do not interfere with active sites or evolutionary conserved regions. Two alternatives should be investigated and presented with regard to present information and distance between fluorophores. The working package is ended with a 3D modeling of the tagging suggestions to further investigate functionality.</p></li> <br />
<li><p><strong>Working Package 2: Decide what fluorescent protein to use</strong><br> <br />
To apply FRET a compatible fluorphore pair is necessary. Also, efficiency and duration in respect to e.g. photonic bleaching should be taken into consideration.</p></li> <br />
<li><p><strong>Working package 3: Find DNA sequence for SAMS-peptide</strong><br> <br />
Beside the two tagging alternative from WP1 a SAMS-peptide will be used. Both ends will be tagged with fluorophores and conformational change should be indicated with FRET-signal during conformational change at phophorylation by active SNF1.</p></li> <br />
<li><p><strong> Working package 4: Decide on plasmid backbone</strong><br><br />
The plasmid containing fusion protein should be able to amplify in E.coli and also contain a strong promoter for protein expression in yeast. Use VectorNTI to find good alternatives and locate RE sites.</p></li> <br />
<li><p><strong>Working package 5: Design primers for fusion PCR</strong><br><br />
When WP 1-4 is finished the primer design can be initiated. Primers should be designed so that they have a high melting temperature, preferably around 60°C, with at least 20 bp homology with each side that should be fused. Primers at utter end of the fusion proteins should include restriction enzyme sites with GGCC repetition at the end. After rigorous proofreading the primers should be ordered together with a primer representing the SAMS peptide.</p></li> <br />
<li><p><strong> Working package 6: Acquire plasmid backbone for transfection with fusion protein</strong><br><br />
Look up how the plasmid backbone from WP4 can be acquired. If it is a common backbone it should be avaible in the plasmid bank at the laboratory, otherwise it should be ordered.</p></li> <br />
<li><p><strong> Working package 7: Perform fusion PCR</strong><br><br />
When the primers from WP5 has arrived the fusion PCR can be initiated. As a template for the protein subunits in SNF1 genomic DNA from yeast should be used, this should be avaible at the laboratory, and as template for fluorescent proteins dried DNA provided by iGEM will be used. Create a PCR mastermix that includes all ingredients except the DNA template. Each fusion is run in five steps according to the manual and each step is analyzed on agar gel so that the correct fragments are produced.</p></li> <br />
<br />
<li><p><strong> Working package 8: Ligate insert into plasmids</strong><br><br />
After WP7 is successful the fusion protein should be inserted into the plasmid backbone. Use restriction enzymes as decided in WP6 and follow ligation protocol. Afterwards the plasmids should be purified with miniprep, they should be cut again and put on a gel to verify that the insert was successful.<br> <br> <br />
<li><p><strong> Working package 9: Amplify transfection plasmids</strong><br><br />
After WP8 the plasmid need to be amplified before being tranfected into yeast. Tranfect E.coli and amplify over night. Purify the amplified plasmids with a miniprep kit.</p></li> <br />
<li><p><strong> Working package 10: Sporulate diploid yeast strains to acquire Snf1 deletion mutants</strong><br><br />
Grow diploid yeast strains with Snf1 deletions on sporulation plats to acquire haploid deletion mutants. Use protocol to clear the population from diploids and unwanted haploids. <br><br> <br />
<li><p><strong> Working package 11: Transfect yeast with tagged fusion protein</strong><br><br />
After WP9-10 the hapoloid yeast deletion strain should be tranfected with the fusion protein plasmid. When the yeast has recovered from transfection the strain should be grown on sucrose overnight to show transfection result.</p></li> <br />
<li><p><strong> Working package 12: Apply FRET and analyze results</strong><br><br />
By using a microscopy with FRET set up the success of previous WP can be concluded from the presence or absence of a FRET signal. Analyze results and make conclusions together with Marcus Wilhelmsson.</p></li> <br />
<li><p><strong>Working package 13:Write report</strong><br><br />
WP 13 is running alongside with the other WPs during the whole project. Initially aims and theory is the major work load and as the project progress results and analysis claims the major part of this WP.</p></li> <br />
<li><p><strong>Working package 14: Apply for funding</strong><br><br />
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.</p></li> <br />
</ul><br />
<br />
<br />
<div id="myTitle">Safety Questions </div> <br />
<br />
<ul id="myList"> <br />
<li><p><strong>Would any of your project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?</strong><br><br />
No, since the yeast strains we work with are derived from harmless bakers yeast an none of the modifications have unknown or harmful consequences for anything else than the organism itself there is no safety issues with the project organism as such. Plasmid amplification in E.coli requiers a selection marker, ampicillin resistance in our case, which pose a safety issue regarding environmental safety but this is easily avoided by following laboration protocols regarding keeping a clean working environment and sterilization of organic waste.</p></li> <br />
<br />
<li><p><strong>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</strong><br><br />
As mentioned above our device contain an ampicillin marker for amplification in E.coli. There could be issues if transfected cells are allowed to be spread outside the controlled laboratory environment. But working with ampicillin resistance as an selection marker is a common procedure and safety protocolls for this kind of work is readilly avaible. This will be regarded when we register our device to iGEM.</p></li> <br />
<br />
<li><p><strong>Is there a local biosafety group, committee, or review board at your institution? </strong><br><br />
No, when working with synthetic biology at Chalmers we follow regulations set up by the Swedish Work Environment Authority. They set up protocols how chemicals and genetically modified material should be treated and what is demanded from an working environment to be permitted with this kind of labor.</p></li> <br />
</ul><br />
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<div class="Title3">Lab Notes</div><br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden
Team:Gothenburg-Sweden
2010-10-03T11:56:23Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
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<p><br />
<strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p> <br />
<br />
<p>We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.<br> <br />
<br> <br />
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.<br> <br />
<br> <br />
The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed. <br> <br />
<br> <br />
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.<br> <br />
<br> <br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Project
Team:Gothenburg-Sweden/Project
2010-10-01T22:38:04Z
<p>Sanli k: </p>
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<div class="Title1">Team</div><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#">Methods</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden">Home</a></li><br />
<li id="active"><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#aim">Aim</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#bkg">Theoretic background</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#method">Methods</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#tech">Techniques</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#snf1">SNF1 &amp; AMPK </a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#bricks">Biobricks</a></li> <br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#app">Possible Applications</a></li> <br />
</ul> <br><br />
<br />
<br />
<div id="myTitle">Project Description </div> <br />
<br />
<p>The conceptual idea is to use the conformational change in the SNF1 protein complex to establish a FRET (Förster Resonance Energy Transfer) system for cellular stress detection. There are two chromophores tagged at appropriate locations and upon undergoing the conformational change, they come in close proximity such that the emission energy from one can excite the other, thus resulting in different emission energy. There are two possibilities to be tested: <br><br>1. To fuse the chromophores to the subunits of the Snf1 complex to build an active readout of the conformational change <br> <br />
2. To fuse the chromophores to a short peptide (SAMS peptide) that can be phosphorylated by Snf1 and is commonly used as a read-out of Snf1 activity. </p> <br> <br />
<br />
<br />
<br />
<ul id="myList"> <li><p> <strong>Working package 1: Find positions for fluorescent markers in SNF1 </strong><br> <br />
For WP1 all present information about the protein complex is utilized, so that the fluorescent tags do not interfere with active sites or evolutionary conserved regions. Two alternatives should be investigated and presented with regard to present information and distance between fluorophores. The working package is ended with a 3D modeling of the tagging suggestions to further investigate functionality.</p></li> <br />
<li><p><strong>Working Package 2: Decide what fluorescent protein to use</strong><br> <br />
To apply FRET a compatible fluorphore pair is necessary. Also, efficiency and duration in respect to e.g. photonic bleaching should be taken into consideration.</p></li> <br />
<li><p><strong>Working package 3: Find DNA sequence for SAMS-peptide</strong><br> <br />
Beside the two tagging alternative from WP1 a SAMS-peptide will be used. Both ends will be tagged with fluorophores and conformational change should be indicated with FRET-signal during conformational change at phophorylation by active SNF1.</p></li> <br />
<li><p><strong> Working package 4: Decide on plasmid backbone</strong><br><br />
The plasmid containing fusion protein should be able to amplify in E.coli and also contain a strong promoter for protein expression in yeast. Use VectorNTI to find good alternatives and locate RE sites.</p></li> <br />
<li><p><strong>Working package 5: Design primers for fusion PCR</strong><br><br />
When WP 1-4 is finished the primer design can be initiated. Primers should be designed so that they have a high melting temperature, preferably around 60°C, with at least 20 bp homology with each side that should be fused. Primers at utter end of the fusion proteins should include restriction enzyme sites with GGCC repetition at the end. After rigorous proofreading the primers should be ordered together with a primer representing the SAMS peptide.</p></li> <br />
<li><p><strong> Working package 6: Acquire plasmid backbone for transfection with fusion protein</strong><br><br />
Look up how the plasmid backbone from WP4 can be acquired. If it is a common backbone it should be avaible in the plasmid bank at the laboratory, otherwise it should be ordered.</p></li> <br />
<li><p><strong> Working package 7: Perform fusion PCR</strong><br><br />
When the primers from WP5 has arrived the fusion PCR can be initiated. As a template for the protein subunits in SNF1 genomic DNA from yeast should be used, this should be avaible at the laboratory, and as template for fluorescent proteins dried DNA provided by iGEM will be used. Create a PCR mastermix that includes all ingredients except the DNA template. Each fusion is run in five steps according to the manual and each step is analyzed on agar gel so that the correct fragments are produced.</p></li> <br />
<br />
<li><p><strong> Working package 8: Ligate insert into plasmids</strong><br><br />
After WP7 is successful the fusion protein should be inserted into the plasmid backbone. Use restriction enzymes as decided in WP6 and follow ligation protocol. Afterwards the plasmids should be purified with miniprep, they should be cut again and put on a gel to verify that the insert was successful.<br> <br> <br />
<li><p><strong> Working package 9: Amplify transfection plasmids</strong><br><br />
After WP8 the plasmid need to be amplified before being tranfected into yeast. Tranfect E.coli and amplify over night. Purify the amplified plasmids with a miniprep kit.</p></li> <br />
<li><p><strong> Working package 10: Sporulate diploid yeast strains to acquire Snf1 deletion mutants</strong><br><br />
Grow diploid yeast strains with Snf1 deletions on sporulation plats to acquire haploid deletion mutants. Use protocol to clear the population from diploids and unwanted haploids. <br><br> <br />
<li><p><strong> Working package 11: Transfect yeast with tagged fusion protein</strong><br><br />
After WP9-10 the hapoloid yeast deletion strain should be tranfected with the fusion protein plasmid. When the yeast has recovered from transfection the strain should be grown on sucrose overnight to show transfection result.</p></li> <br />
<li><p><strong> Working package 12: Apply FRET and analyze results</strong><br><br />
By using a microscopy with FRET set up the success of previous WP can be concluded from the presence or absence of a FRET signal. Analyze results and make conclusions together with Marcus Wilhelmsson.</p></li> <br />
<li><p><strong>Working package 13:Write report</strong><br><br />
WP 13 is running alongside with the other WPs during the whole project. Initially aims and theory is the major work load and as the project progress results and analysis claims the major part of this WP.</p></li> <br />
<li><p><strong>Working package 14: Apply for funding</strong><br><br />
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.</p></li> <br />
</ul><br />
<br />
<br />
<div id="myTitle">Safety Questions </div> <br />
<br />
<ul id="myList"> <br />
<li><p><strong>Would any of your project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety?</strong><br><br />
No, since the yeast strains we work with are derived from harmless bakers yeast an none of the modifications have unknown or harmful consequences for anything else than the organism itself there is no safety issues with the project organism as such. Plasmid amplification in E.coli requiers a selection marker, ampicillin resistance in our case, which pose a safety issue regarding environmental safety but this is easily avoided by following laboration protocols regarding keeping a clean working environment and sterilization of organic waste.</p></li> <br />
<br />
<li><p><strong>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</strong><br><br />
As mentioned above our device contain an ampicillin marker for amplification in E.coli. There could be issues if transfected cells are allowed to be spread outside the controlled laboratory environment. But working with ampicillin resistance as an selection marker is a common procedure and safety protocolls for this kind of work is readilly avaible. This will be regarded when we register our device to iGEM.</p></li> <br />
<br />
<li><p><strong>Is there a local biosafety group, committee, or review board at your institution? </strong><br><br />
No, when working with synthetic biology at Chalmers we follow regulations set up by the Swedish Work Environment Authority. They set up protocols how chemicals and genetically modified material should be treated and what is demanded from an working environment to be permitted with this kind of labor.</p></li> <br />
</ul><br />
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<div class="Title3">Lab Notes</div><br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Sponsors
Team:Gothenburg-Sweden/Sponsors
2010-10-01T21:43:44Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
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<a href="#">What's all about?</a><br />
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<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
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<br />
<p>We are currently looking for an official corporate sponsor that is interested in the field of synthetic biology and the major medical applications our technology could lead to. We have good experience of collaborating with the industry, and if you choose to partner up with us we can assure you that your company will have great international exposure during the iGEM contest. Please contact us at burmank@student.chalmers.se and we can discuss the terms of our companionship.</p><br />
<br><br><br><br><br><br><br><br><br><br><br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Sponsors
Team:Gothenburg-Sweden/Sponsors
2010-10-01T21:42:57Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
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<a href="#"> Methods</a><br />
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<br><br />
<p>We are currently looking for an official corporate sponsor that is interested in the field of synthetic biology and the major medical applications our technology could lead to. We have good experience of collaborating with the industry, and if you choose to partner up with us we can assure you that your company will have great international exposure during the iGEM contest. Please contact us at burmank@student.chalmers.se and we can discuss the terms of our companionship.</p><br />
<br><br><br><br><br><br><br><br><br><br><br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden
Team:Gothenburg-Sweden
2010-10-01T14:00:33Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#">Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
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<p><br />
<strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p> <br />
<br />
<p>We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.<br> <br />
<br> <br />
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.<br> <br />
<br> <br />
The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed. <br> <br />
<br> <br />
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.<br> <br />
<br> <br />
<br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br></p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/About_us
Team:Gothenburg-Sweden/About us
2010-10-01T13:59:14Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a><br />
<br />
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<br />
<div class="Title1">FUSS</div><br />
<br />
<div class="left_grad"><br />
<div class="categories"><br />
<br />
<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<div id="Title2"><ul id="sddm"><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden">Home</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
</div><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
</ul><br />
<div style="clear:both"></div></div><br />
<div id="text"><a name="intro"></a><br />
<p> We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines the cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing a reporter mechanism for cellular stress in yeast by tagging protein and peptides affiliated with the stress activated SNF1 complex with fluorescent markers. </p> <br />
<br />
<br />
<br />
<br />
<div id="myTitle">Chalmers University of Technology <a name="cth"></a> </div> <br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/a/a5/Chalmers_c_logo.jpg" width="133" height="152" align="left"></div><br />
<br />
<p><br />
<strong>Chalmers in central Göteborg on the Swedish West Coast</strong></p> <br />
<br />
<p>Chalmers is a university of technology in which research and teaching are conducted on a broad front within technology, natural science and architecture. Our inspiration lies in the joy of discovery and the desire to learn. Underlying everything we do is a wish to contribute to sustainable development both in Sweden and world-wide.<br> <br />
<br> <br />
Our research work deals ultimately with improving people’s conditions, and we often cross traditional boundaries in order to solve the problems of the future. Chalmers has become strong within several areas of science, and some of our research leads its field internationally. We wish in particular to develop and strengthen our research in the fields of bioscience, information technology, environmental science and nanotechnology.<br> <br />
<br> <br />
Chalmers is a pioneer in Europe in the field of start-up companies, and has built up an efficient system of innovation in order to find applications in research into the formation of companies. <br> <br />
<br> <br />
Chalmers has great aspirations to develop continually by opening doors to the outside world and by thinking innovatively. We have a close and productive collaboration with business and society at large. In this way we reinforce our own development, at the same time as we contribute to the growth of society.<br> <br />
<br> <br />
Chalmers was founded in 1829 as a result of a donation from the director of the Swedish East India Company, William Chalmers. His motto, and ours, is:<br> <br />
<br> <br />
Avancez!</p> <br />
<br />
<br />
<br />
<br />
<div id="myTitle">Supervisors<a name="svs"></a></div> <br />
<br />
<div id="team"><br />
<td><table width="560" border="1"> <br />
<tr> <br />
<td width="145" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/eb/Goutham.jpg" alt="" width="133" height="200" class="img1" /></span></td> <br />
<td width="174" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"> <br />
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<td><span class="style9">Goutham Vemuri</span></td> <br />
</tr> <br />
<tr> <br />
<td><span class="style11">supervisor</span></td> <br />
</tr> <br />
</table></td> <br />
</tr> <br />
<tr> <br />
<td valign="top"><p>&nbsp;</p> <br />
</td> <br />
<td width="214" valign="top"><p class="style5">&nbsp;</p> <br />
</table> <br />
<p>&nbsp;</p> <br />
<table width="560" border="1"> <br />
<tr> <br />
<td width="145" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/3/32/Per.jpg" alt="" width="133" height="200" class="img1" /></span></td> <br />
<td width="174" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"> <br />
<tr> <br />
<td><span class="style9">Per Sunnerhagen </span></td> <br />
</tr> <br />
<tr> <br />
<td><span class="style11">supervisor</span></td> <br />
</tr> <br />
</table></td> <br />
</tr> <br />
<tr> <br />
<td valign="top"><p>&nbsp;</p></td> <br />
<td width="214" valign="top"><p class="style5">&nbsp;</p> <br />
</table> <br />
<p>&nbsp;</p> <br />
<table width="560" border="1"> <br />
<tr> <br />
<td width="145" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/8/88/Markus.jpg" alt="" width="133" height="200" class="img1" /></span></td> <br />
<td width="169" height="59" valign="top"><table width="172" border="5" cellpadding="0" cellspacing="0"> <br />
<tr> <br />
<td width="136"><span class="style9">Marcus Wilhelmsson </span></td> <br />
</tr> <br />
<tr> <br />
<td><span class="style11">supervisor</span></td> <br />
</tr> <br />
</table></td> <br />
</tr> <br />
<tr> <br />
<td valign="top"><p>&nbsp;</p></td> <br />
<td width="219" valign="top"><p class="style5">&nbsp;</p> <br />
</table> <br />
<td>&nbsp;</td> <br />
</tr> <br />
<tr></tr> <br />
<tr> <br />
<td>&nbsp;</td> <br />
</tr> <br />
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<br />
<br />
<br />
<br />
<div id="myTitle">Students<a name="sts"></a></div> <br />
<br />
<div id="team"><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/d/d0/Malin.jpg" alt="" width="133" height="200" class="img1" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td><span class="style9">Malin Linden</span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
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</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="363" height="150" border="1"><br />
<tr><br />
<td width="147"><span class="style4">Age:</span></td><br />
<td width="158"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">Exchange year at University of Minnesota, MS Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"> <br />
<tr> <br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/7/7f/Katarina.jpg" alt="" width="133" height="200" class="img1" /></span></td> <br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"> <br />
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<td><span class="style9">Katarina Risö </span></td> <br />
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<tr> <br />
<td><span class="style11">student</span></td> <br />
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<td width="237">&nbsp;</td> <br />
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<tr> <br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Chemical Engineering</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Chemistry and Biosciences</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table><br />
</td> <br />
</tr> <br />
</table> <br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/a/a7/Peidi.jpg" alt="" width="133" height="200" class="img1" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td><span class="style9">Peidi Liu </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Chinese</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">Electronic Engineering</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics </span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/8/80/Adnan.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
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<td><span class="style9">Adnan Kadic</span></td><br />
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<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Bosnian</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Chemistry and Biosciences </span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
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<p>&nbsp;</p> <br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/ea/Lokesh.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
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<td width="180"><span class="style9">Lokeshwaran Manoharan </span></td><br />
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<tr><br />
<td><span class="style11">student</span></td><br />
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<td width="124">&nbsp;</td><br />
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<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">23</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Indian</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B.Tech Ind Biotechnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/0/01/Kemal.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Kemal Sanli </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Turkish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Molecular Biology and Genetics </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/4/40/Karl.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Karl Almén Burman </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">23</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotechnology </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Biotechnology</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/e8/Julia.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Julia Fransson </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">22</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotechnology </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">Exchange year through Unitech International</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<p>&nbsp;</p> <br />
</td><br />
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<tr><td>&nbsp;</td> <br />
</tr><tr><td>&nbsp;</td> <br />
</tr> <br />
</div><br />
<br />
</div> <br />
</div><br />
<div id="right"><br />
<div class="Title3">Lab Notes</div><br />
<br />
<br />
<div id="lab"><br />
<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
<br />
<br />
<br />
</div> <br />
<div class="Title3">News</div><br />
<div id="news"><br />
<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
</div> <br />
<br />
<br />
<br />
</div><br />
<br />
<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
<br />
</div><br />
</div></div> <br />
</body><br />
</html></div>
Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden
Team:Gothenburg-Sweden
2010-10-01T13:56:17Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
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<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/a/a5/Chalmers_c_logo.jpg" width="133" height="152" align="left"></div><br />
<br />
<p><br />
<strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p> <br />
<br />
<p>We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.<br> <br />
<br> <br />
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.<br> <br />
<br> <br />
The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed. <br> <br />
<br> <br />
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.<br> <br />
<br> <br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<br />
<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br><br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden
Team:Gothenburg-Sweden
2010-10-01T13:55:52Z
<p>Sanli k: </p>
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<br />
<br />
<div class="Title1">Team</div><br />
<div class="left_grad"><br />
<div class="categories"><br />
<br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a><br />
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<div class="Title1">FUSS</div><br />
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<div class="left_grad"><br />
<div class="categories"><br />
<br />
<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#">Methods</a><br />
<a href="#">Preliminary Results</a><br />
<br />
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<li id="active"><a href="https://2010.igem.org/Team:Gothenburg-Sweden">Home</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<div id="m1" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
</div><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/a/a5/Chalmers_c_logo.jpg" width="133" height="152" align="left"></div><br />
<br><br />
<p><br />
<strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p> <br />
<br />
<p>We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.<br> <br />
<br> <br />
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.<br> <br />
<br> <br />
The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed. <br> <br />
<br> <br />
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.<br> <br />
<br> <br />
<br />
</div> </div><br />
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<div id="right"><br />
<div class="Title3">Lab Notes</div><br />
<br />
<br />
<div id="lab"><br />
<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
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<div class="Title3">News</div><br />
<div id="news"><br />
<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br><br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
<br />
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</html></div>
Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden
Team:Gothenburg-Sweden
2010-10-01T13:55:21Z
<p>Sanli k: </p>
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<div class="Title1">Team</div><br />
<div class="left_grad"><br />
<div class="categories"><br />
<br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a><br />
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<div class="Title1">FUSS</div><br />
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<br />
<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#">Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us" <br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<p><br />
<strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p> <br />
<br />
<p>We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.<br> <br />
<br> <br />
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.<br> <br />
<br> <br />
The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed. <br> <br />
<br> <br />
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.<br> <br />
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<div class="Title3">Lab Notes</div><br />
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<p>11</p><br />
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<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<div class="read"><a href="#">read more</a></div><br />
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<div class="Title3">News</div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br><br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Results
Team:Gothenburg-Sweden/Results
2010-10-01T13:54:32Z
<p>Sanli k: </p>
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<div class="Title1">Team</div><br />
<div class="left_grad"><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<div id="Title2"><ul id="sddm"><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden">Home</a></li><br />
<li id="active"><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
</div><br />
</li><br />
<br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
</ul><br />
<div style="clear:both"></div></div><br />
<div id="text"><br />
<br />
<br />
<p>coming soon...</p><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> <br> <br />
</div> </div><br />
<br />
<br />
<br />
<div id="right"><br />
<div class="Title3">Lab Notes</div><br />
<br />
<br />
<div id="lab"><br />
<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
<br />
<br />
<br />
</div> <br />
<div class="Title3">News</div><br />
<div id="news"><br />
<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br></p><br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
</div> <br />
<br />
<br />
<br />
</div><br />
<br />
<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
<br />
</div><br />
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</body><br />
</html></div>
Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Results
Team:Gothenburg-Sweden/Results
2010-10-01T13:53:44Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
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<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
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<a href="#">Chalmers University of Technology</a><br />
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<div style="clear:both"></div></div><br />
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<br />
<br />
<p>coming soon...</p><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
</div> </div><br />
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<div class="Title3">Lab Notes</div><br />
<br />
<br />
<div id="lab"><br />
<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
<br />
<br />
<br />
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<div class="Title3">News</div><br />
<div id="news"><br />
<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br><br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
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<br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden
Team:Gothenburg-Sweden
2010-10-01T13:53:18Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
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<a href="#">What's all about?</a><br />
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<a href="#">Chalmers University of Technology</a><br />
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<strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p> <br />
<br />
<p>We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.<br> <br />
<br> <br />
The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.<br> <br />
<br> <br />
The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed. <br> <br />
<br> <br />
As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.<br> <br />
<br> <br />
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<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<div class="read"><a href="#">read more</a></div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br><br> </p><br />
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<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
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Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/About_us
Team:Gothenburg-Sweden/About us
2010-10-01T13:51:04Z
<p>Sanli k: </p>
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a><br />
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<br />
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<br />
<a href="#">What's all about?</a><br />
<a href="#">Background</a><br />
<a href="#">Protein Fusion</a><br />
<a href="#"> Methods</a><br />
<a href="#">Preliminary Results</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project" <br />
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<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a><br />
<a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a><br />
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<a href="#">Chalmers University of Technology</a><br />
<a href="#">Supervisors</a><br />
<a href="#">Students</a><br />
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<li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li><br />
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<div id="text"><a name="intro"></a><br />
<p> We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines the cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing a reporter mechanism for cellular stress in yeast by tagging protein and peptides affiliated with the stress activated SNF1 complex with fluorescent markers. </p> <br />
<br />
<br />
<br />
<br />
<div id="myTitle">Chalmers University of Technology <a name="cth"></a> </div> <br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/a/a5/Chalmers_c_logo.jpg" width="133" height="152" align="left"></div><br />
<br />
<p><br />
<strong>Chalmers in central Göteborg on the Swedish West Coast</strong></p> <br />
<br />
<p>Chalmers is a university of technology in which research and teaching are conducted on a broad front within technology, natural science and architecture. Our inspiration lies in the joy of discovery and the desire to learn. Underlying everything we do is a wish to contribute to sustainable development both in Sweden and world-wide.<br> <br />
<br> <br />
Our research work deals ultimately with improving people’s conditions, and we often cross traditional boundaries in order to solve the problems of the future. Chalmers has become strong within several areas of science, and some of our research leads its field internationally. We wish in particular to develop and strengthen our research in the fields of bioscience, information technology, environmental science and nanotechnology.<br> <br />
<br> <br />
Chalmers is a pioneer in Europe in the field of start-up companies, and has built up an efficient system of innovation in order to find applications in research into the formation of companies. <br> <br />
<br> <br />
Chalmers has great aspirations to develop continually by opening doors to the outside world and by thinking innovatively. We have a close and productive collaboration with business and society at large. In this way we reinforce our own development, at the same time as we contribute to the growth of society.<br> <br />
<br> <br />
Chalmers was founded in 1829 as a result of a donation from the director of the Swedish East India Company, William Chalmers. His motto, and ours, is:<br> <br />
<br> <br />
Avancez!</p> <br />
<br />
<br />
<br />
<br />
<div id="myTitle">Supervisors<a name="svs"></a></div> <br />
<br />
<div id="team"><br />
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<td><span class="style9">Per Sunnerhagen </span></td> <br />
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<td width="136"><span class="style9">Marcus Wilhelmsson </span></td> <br />
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<div id="myTitle">Students<a name="sts"></a></div> <br />
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<td width="237">&nbsp;</td><br />
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<td width="147"><span class="style4">Age:</span></td><br />
<td width="158"><span class="style5">24</span></td><br />
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<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">Exchange year at University of Minnesota, MS Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"> <br />
<tr> <br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/7/7f/Katarina.jpg" alt="" width="133" height="200" class="img1" /></span></td> <br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"> <br />
<tr> <br />
<td><span class="style9">Katarina Risö </span></td> <br />
</tr> <br />
<tr> <br />
<td><span class="style11">student</span></td> <br />
</tr> <br />
</table></td> <br />
<td width="237">&nbsp;</td> <br />
</tr> <br />
<tr> <br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Chemical Engineering</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Chemistry and Biosciences</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table><br />
</td> <br />
</tr> <br />
</table> <br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/a/a7/Peidi.jpg" alt="" width="133" height="200" class="img1" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td><span class="style9">Peidi Liu </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Chinese</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">Electronic Engineering</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics </span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/8/80/Adnan.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="151" height="59" valign="top"><table width="150" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td><span class="style9">Adnan Kadic</span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="237">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Bosnian</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotecnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Chemistry and Biosciences </span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/ea/Lokesh.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="221" height="59" valign="top"><table width="216" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="180"><span class="style9">Lokeshwaran Manoharan </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="124">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="140"><span class="style4">Age:</span></td><br />
<td width="170"><span class="style5">23</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Indian</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B.Tech Ind Biotechnology</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/0/01/Kemal.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Kemal Sanli </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">24</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Turkish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Molecular Biology and Genetics </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Bioinformatics</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/4/40/Karl.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Karl Almén Burman </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">23</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotechnology </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">MS Biotechnology</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<table width="560" border="1"><br />
<tr><br />
<td width="150" rowspan="2" valign="top"><span class="style8"><img src="https://static.igem.org/mediawiki/2010/e/e8/Julia.jpg" alt="" width="133" height="200" class="style5" /></span></td><br />
<td width="207" height="59" valign="top"><table width="186" border="5" cellpadding="0" cellspacing="0"><br />
<tr><br />
<td width="150"><span class="style9">Julia Fransson </span></td><br />
</tr><br />
<tr><br />
<td><span class="style11">student</span></td><br />
</tr><br />
</table></td><br />
<td width="161">&nbsp;</td><br />
</tr><br />
<tr><br />
<td colspan="2" valign="top"><table width="368" height="150" border="1"><br />
<tr><br />
<td width="133"><span class="style4">Age:</span></td><br />
<td width="177"><span class="style5">22</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Nationality:</strong></td><br />
<td><span class="style5">Swedish</span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Academic background:</strong></td><br />
<td><span class="style5">B sc. Biotechnology </span></td><br />
</tr><br />
<tr><br />
<td><strong class="style5">Current studies:</strong></td><br />
<td><span class="style5">Exchange year through Unitech International</span></td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td>&nbsp;</td><br />
</tr><br />
</table></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p> <br />
<p>&nbsp;</p> <br />
</td><br />
</tr><br />
<tr><td>&nbsp;</td> <br />
</tr><tr><td>&nbsp;</td> <br />
</tr> <br />
</div><br />
<br />
</div> <br />
</div><br />
<div id="right"><br />
<div class="Title3">Lab Notes</div><br />
<br />
<br />
<div id="lab"><br />
<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
</div><br />
<br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
<br />
<br />
<br />
</div> <br />
<div class="Title3">News</div><br />
<div id="news"><br />
<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
<br> </p><br />
<div class="read"><a href="#">read more</a></div><br />
<div style="clear: both"></div><br />
</div> <br />
<br />
<br />
<br />
</div><br />
<br />
<div id="myfooter"> <p> <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p><br />
<br />
</div><br />
</div></div> <br />
</body><br />
</html></div>
Sanli k
http://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note
Team:Gothenburg-Sweden/Lab Note
2010-10-01T13:50:34Z
<p>Sanli k: </p>
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<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/f/fe/Gfp.png" width="40" height="40" align="left"></div> <strong><p>Fluorescent Proteins:</strong><br> Green Fluorescent Protein <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p><br> <br />
<br />
<div class="logo"> <img src=" https://static.igem.org/mediawiki/2010/a/a9/Plasmid.png" width="40" height="40" align="left"></div><p><strong>Plasmid backbone:</strong> <br><br />
pSP-GM1 <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#plasmid">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/7/74/Snf1Com.png" width="40" height="40" align="left"></div><p> <strong>SNF1 (modified subunits):</strong><br><br />
Snf1 (alfa), Snf4 (gamma) <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#SNF1">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/4/46/FpPos.png" width="40" height="40" align="left"></div> <p> <strong>FP positions:</strong> <br><br />
N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#position">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/e/e0/PrDesign.png" width="40" height="40" align="left"></div> <p> <strong>Primer design:</strong><br> <br />
Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#primer">(read more)</a></p> <br> <br />
<br />
<br />
<br />
<div id="myTitle">Lab work by date </div> <br />
<ul><li><p> <strong>2010-08-11</strong><br> <br />
Malin and Katarina<br> <br />
<br> <br />
The PCR reaction from yesterday was checked on a gel. α should be around 1500 bp but only a band around 750 bp was seen. We had a long meeting with supervisor Per about PCR reactions and the next couple of weeks in the lab. <br> <br />
<br> <br />
Karl and Kemal <br> <br />
<br> <br />
Ran PCR reaction α with different settings. <br> <br />
<strong>α1: </strong>regular settings<br> <br />
<strong>α2: </strong>10 x diluted template 2<br> <br />
<strong>α3: </strong>increased annealing time from 30 s to 60 s<br> <br />
<strong>α4: </strong>decreased annealing temperature<br> <br />
<br> <br />
After PCR, the products were run on a gel. α1, α3 and α 4 had a band around 750 bp but nothing on α2. </p></li><br> <br />
<br> <br />
<li><p> <strong>2010-08-10</strong><br> <br />
Peidi and Lokesh<br> <br />
<br> <br />
Checked the transformation plates but nothing had grown. Prepared PCR reaction Γ. <br> <br />
<br> <br />
Katarina and Kemal<br> <br />
<br> <br />
PCR product Γ was verified on a 0.7 % agarose gel. There was a band around 750 bp as expected and it was purified using the regular protocol. A new phusion mastermix was made and PCR reaction α was run with Γ as one of the template. Regular phusion PCR settings was used. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-08-09</strong><br> <br />
Malin and Kemal<br> <br />
<br> <br />
Before checking if the ligation worked we need to transform and amplify in E.coli. Then we can miniprep the ligated plasmid and check on a gel. After that we can digest plasmid with PacI and BcuI and ligate the digested IV. Then another round of transformation and miniprep before we can transform yeast. <br> <br />
<br> <br />
Karl and Lokesh<br> <br />
<br> <br />
Transformed competent E.coli with plasmid ligated with III by heat shock. The cells were incubated at 37 °C over night. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-07</strong><br> <br />
Lokesh<br> <br />
<br> <br />
Moved ligation tube to freezer. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-06</strong><br> <br />
Malin, Julia and Lokesh<br> <br />
<br> <br />
III and IV were digested 3x protocol and were then put on a 1 % agarose gel together with Γ both for verification and purification. Correct bands were seen for purification except for Γ which was empty. In the verification lanes the digested PCR products were to light to be seen but they were seen in purification lanes. IIId and IVd were purified. <br> <br />
<br> <br />
<strong>IIId: </strong>7.8 ng/µl<br> <br />
<strong>IVd: </strong>11.7ng/µl<br> <br />
<br> <br />
IIId will now be ligated with the cut plasmid (BamHI and HindIII). Ligation performed according to protocol with 156 ng insert and 52 ng plasmid. Ligation was incubated over night in fridge at 4-16° C. <br> <br />
<br> <br />
All 4 sporulation plates were checked but still no tetrads visible. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-05</strong><br> <br />
Malin and Julia<br> <br />
<br> <br />
III and IV were digested using the regular protocol. <br> <br />
<strong>III: </strong>HindIII and BamHI works with Buffer B<br> <br />
<strong>IV: </strong>PacI and BcuI works with Buffer M<br> <br />
<br> <br />
A 1% gel was run with uncut and digested III and IV, control, I and Β. I worked well and will be purified. The Β lane showed no bands. None of the digested products were visible on the gel so a new gel was run trying to verify this result. The control shows a band around 1700 which is weird. This band is also visible on the III uncut but together with a band around 750 bp. No bands at the IV which is very strange since this PCR product has been verified before. Have something gone wrong with the purification? <br> <br />
<br> <br />
As a result PCR reactions III and IV will be redone. New PCR reactions of Γ and α were also performed in the same block. <br> <br />
<br> <br />
Karl and Kemal<br> <br />
<br> <br />
Reactions of Β were made. Β1 is a continuation of the 100 cycle PCR this time with primers. Β2 uses a megaprimer approach. <br> <br />
<br> <br />
All the PCR products were run on a 1 % agarose gel. III, IV and Γ were alright. α and Β2 showed a band around 750 bp. <br> <br />
<strong>III: </strong>531.3 ng/µl<br> <br />
<strong>IV: </strong>263.9 ng/µl<br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-04</strong><br> <br />
Julia and Karl<br> <br />
<br> <br />
PCR products Β were loaded on a 0.7 % gel. No correct bands, only amplified templates around 750 bp and something else around 500 bp. <br> <br />
<br> <br />
PCR of SAMS will also be redone from scratch. PCR reactions 1, 2 and C are done, then loaded on a gel (2 % gel and low range ladder) and purified. <br> <br />
<strong>1: </strong>8.2 ng/µl<br> <br />
<strong>2: </strong>7.8 ng/µl<br> <br />
<strong>C: </strong>4.9 ng/µl<br> <br />
Samples diluted to 1 ng/µl. <br> <br />
<br> <br />
PCR of Β was done again but this time in two steps and with the Finnzymes Tm which corresponds to an annealing temp at 85 °C. Firstly 10 cycles without primers were performed. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
Second round of PCR with primers were performed. Then new PCR reactions amplifying III and IV were done and also reaction I. <br> <br />
<strong>III: </strong>572 ng/µl<br> <br />
<strong>IV: </strong>660 ng/µl<br> <br />
<br> <br />
The sporplates were checked and very few tetrads were seen. <br> <br />
<br> <br />
Β samples were put on a gel but no correct bands visible. We suspect that the reverse primer is attaching in the wrong place since YFP and CFP is so similar which then would lead to 750 bp fragment. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-03</strong><br> <br />
Malin and Lokesh<br> <br />
<br> <br />
PCR products II, III and IV were verified and purified from a 1 % agarose gel. III did not work and was not purified. <br> <br />
<br> <br />
Concentrations: <br> <br />
<strong>II: </strong>6.2 ng/µl <br> <br />
<strong>IV: </strong>5.6 ng/µl<br> <br />
II and IV were diluted to 1 ng/µl. <br> <br />
<br> <br />
The new sporplates were examined but no tetrads visible. To check further the cells were stained with DAPI and examined in fluorescence microscope. Still no success. <br> <br />
<br> <br />
New PCR reactions were prepared. III was redone and Β was also done. Same settings as yesterday. <br> <br />
<br> <br />
Kemal and Karl<br> <br />
<br> <br />
PCR products were loaded on a 1% agarose gel for verification and purification. III worked this time and was purified from the gel using the protocol from QIAquick Gel Extraction Kit. <br> <br />
<strong>III: </strong>6.4 ng/µl<br> <br />
<br> <br />
New PCR of Β was run with two different block settings. <br> <br />
<strong>Block A: </strong>increased annealing time to 1 min. <br> <br />
<strong>Block B: </strong>increased annealing time to 58 °C. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-02</strong><br> <br />
Kemal, Lokesh and Karl<br> <br />
<br> <br />
PCR products A, B and 3 were loaded on a gel and then purified from the gel using the protocol from QIAquick Gel Extraction Kit. α and IV were also run on a gel but only showed strong bands corresponding to amplified templates. On the α lane however a very light band was seen at the correct length. This will be further investigated. <br> <br />
<br> <br />
Malin and Julia<br> <br />
<br> <br />
The purified PCR products were measured with nanodrop: <br> <br />
<strong>A1: </strong>11.8 ng/µl<br> <br />
<strong>A2: </strong>10.2 ng/µl<br> <br />
<strong>B1: </strong>9.6 ng/µl<br> <br />
<strong>B2: </strong>17.1 ng/µl<br> <br />
<strong>31: </strong>15.0 ng/µl<br> <br />
<strong>32: </strong>16.3 ng/µl<br> <br />
<br> <br />
A, B and 3 were diluted to 1 ng/µl. <br> <br />
<br> <br />
PCR products 4 and 5 were loaded on a 1 % agarose gel together with IV that should be controlled. PCR products 4 and 5 were correct so they were purified from the gel using the protocol from QIAquick Gel Extraction Kit. IV however was not correct. Only a band around 750 bp was visible. <br> <br />
<br> <br />
Concentrations: <br> <br />
<strong>4: </strong>10.8 ng/µl<br> <br />
<strong>5: </strong>10.4 ng/µl<br> <br />
4-5 were diluted to 1 ng/µl. <br> <br />
New PCR reactions were started (II, III and IV). PCR settings that works well with the Phusion enzyme was set: <br> <br />
Initial denaturation: 98 °C 3 min<br> <br />
Denaturation: 98 °C 10 s<br> <br />
Annealing: 55 °C 30 s<br> <br />
Elongation: 72 °C 1.5 min+ 1s/cycle<br> <br />
30 cycles<br> <br />
Final extension: 72 °C 10 min<br> <br />
4°C forever <br> <br />
<br> <br />
<strong>2010-07-30</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
The concentration of the purified plasmids were measured with nanodrop: <br> <br />
<strong>P1: </strong>1.7 ng/µl<br> <br />
<strong>P2: </strong>3.5 ng/µl<br> <br />
<strong>P3: </strong>25.9 ng/µl<br> <br />
<strong>P4: </strong>42.8 ng/µl<br> <br />
This is clear evidence that water should be used for eluation in the future! <br> <br />
<br> <br />
To make sure that we have the right products after purification a 1 % agarose gel was run with fastruler DNA ladder. The gel verified the plasmid size though the bands were light. <br> <br />
<br> <br />
PCR product III was evaporated since it was so dilute. New concentration: <br> <br />
<strong>IIId: </strong>35 ng/µL<br> <br />
<br> <br />
The digested PCR product III and the digested plasmid were ligated with T4 ligase. The DNA concentration in total should not exceed 1 µg. The ratio insert:vector should be 3:1. <br> <br />
<br> <br />
<strong>Ligation protocol: </strong><br> <br />
150 ng insert<br> <br />
50 ng vector<br> <br />
3 µl 10x T4 buffer<br> <br />
2.5 µl (2.5 U, between 1-5 U) T4 ligase<br> <br />
Adjust volume with water up to 30 µl. Mix and incubate in 4-16 °C (refrigerator) overnight. <br> <br />
The newly plated sporplates were moved to 30°C. The old sporplates were again studied in the microscope and no tetrads were seen. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
Since we have had a lot of trouble with the longer fusions we will start over from scratch with a new high fidelity polymerase called “Finnzumes Phusion”. New PCR reactions were run (A, B, 3, 4 and 5). IV and α were also performed using alternative settings (2 different PCR reactions described before) but with “old” templates. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-29</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
PCR product III is the final fusion protein where Snf4 is tagged with yfp. To be able to insert the construct into the plasmid we must digest it. <br> <br />
<br> <br />
Digestion: Mix DNA, buffer and water. Last add enzyme and mix. Incubate at 37° C for 1 hour. <br> <br />
1 µg DNA<br> <br />
2.5 µl Buffer<br> <br />
1 U Enzyme<br> <br />
Up to 25 µl H2O<br> <br />
For PCR product III, the restriction enzymes BamHI and HindIII are used together with buffer B. After digestion (the protocol was scaled 3x) the sample was loaded on a 1 % agarose gel in 6 lanes with 1 kb ladder. The bands were cut out and weighed. The samples were purified from the gel using the protocol from QIAquick Gel Extraction Kit. The DNA was eluated with water. <br> <br />
<br> <br />
Concentration: <br> <br />
<strong>IIId: </strong>5 ng/µl (Total product 160 µl= 800 ng) <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
The plasmids were also digested with BamHI and HindIII according to the same protocol as above. P1 and P2 were eluated with buffer EB and P3 and P4 with water. <br> <br />
<br> <br />
2 new sporulation plates were prepared by plating the diploid yeast cells on spore plates and kept at room temperature. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-28</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
The PCR products were run on a 1 % gel with 1 kb ladder. Γ may have worked. α and Β had a band around 750 bp and a weird light band around 500 bp. They obviously did not work. IV did not work at all. <br> <br />
<br> <br />
The sporulation plates were checked for spores. Cells were studied under a microscope. No tetrads visible. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-07-27</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
A 1.5 % gel was run with all of the fragments around 700 bp to control the validity of the PCR reactions. A low range ladder was used. PCR products 1-5 seems good. The results from yesterday were confirmed. <br> <br />
<br> <br />
A new PCR design was made with α, Β, Γ and IV. PCR was done in two steps to increase the chance of success for longer fusions. <br> <br />
1. 7 cycles with short annealing time. <br> <br />
2. 20 cycles with long annealing time (all other settings kept standard). <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-26</strong><br> <br />
<br> <br />
A new experiment were designed called Γ which is the same as I but with longer annealing region. Also an experiment called Δ were designed which corresponds to II. <br> <br />
<br> <br />
Julia and Katarina<br> <br />
<br> <br />
PCR reactions were run with samples of 4, IV and Γ. IV was tried with two different PCR settings: one regular and one with two runs of PCR, the first without primers and only 5 cycles and the second one with regular settings. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
The PCR products were controlled on a 0.7% agarose gel. Only 4 worked. Γ was empty and IV had bands in the wrong place (amplified template). <br> <br />
<strong>4: </strong>534 ng/ µl </p></li> <br />
<li><p> <strong>2010-07-22</strong><br> <br />
Karl, Lokesh and Kemal<br />
</br></br> <br />
A new mastermix was prepared using a different enzyme. Then the experiments from yesterday was redone using regular PCR settings: <br> <br />
A, 5, 2, I<br> <br />
Then some more experiments of IV was redone: <br> <br />
2a, 2b and 2c<br> <br />
The new primer 14 had arrived so PCR reaction 4 was also redone. <br> <br />
<br> <br />
All the PCR products were run on a 0.7 % agarose gel with GeneRuler 1 kb DNA ladder. The results were inconclusive. None of the expected bands were received. At this point we are not sure what to do since none of the PCR reactions worked. <br> <br />
<br> <br />
</p> <br />
</li> <br />
<li> <p> <strong>2010-07-21</strong><br> <br />
Malin, Julia, Karl and Lokesh<br> <br />
<br> <br />
<strong>Verification of the PCR products </strong>α1, α2, α3, α4, α5 and αK by running on a thin 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder). <br> <br />
<br> <br />
The gel didn’t show the expected band lengths. A band around 700 bp could be seen, most strongly for α4 and α5. We suspect that there is something wrong with the ladder because we get inconclusive results. Since none of the PCR reactions for the last couple of days have worked we will redo the templates of α and IV: I, 2, 5 and A. These PCR reactions have worked before. <br> <br />
<br> <br />
<strong>Expected fragment length: </strong><br> <br />
A.2: 1938 bp<br> <br />
5.2: 757 bp<br> <br />
2.2: 762 bp<br> <br />
I.2: 768 bp<br> <br />
<br> <br />
The PCR products were run on a thin 0.7 % agarose gel. We used both the FastRuler High Range Ladder and the GeneRuler 1 kb DNA ladder. 5.2 and 2.2 showed the expected bands around 750 bp. I.2 and A.2 did not work at all, nothing visible on the gel. We also run some of the old PCR products to see what the result would be using a new ladder. We run α2, α5, 4c (IV) and 4c*(IV). Here some template amplifications were visible but sadly none of the expected fragment lengths. Both ladders showed the same result so there is probably nothing wrong with the GeneRuler ladder except that it is not showing strong color. <br></p></li> <br />
<li><p><strong>2010-07-20</strong><br> <br />
Malin, Julia, Lokesh and Karl<br> <br />
<br> <br />
<strong>Verification of yesterdays PCR reactions </strong>by running on a 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder) <br> <br />
<br> <br />
None of the reactions worked. <br> <br />
<br> <br />
Gel 1 (Block A): Nothing visible on gel, ladders only very light so these samples were run on a new 0.7 % agarose gel but this time thinner to get better resulotion (0.58 g agarose + 80 ml TBE). This time no expected fragments were seen, but bands corresponding to primer dimers were clearly seen. <br> <br />
Gel 2 (Block B): A few small bands were seen but no bands of expected length. <br> <br />
<br> <br />
We decided to stop working on IV until proper feedback on the experimental setup was received. Instead we continued with α.<br> <br />
<br> <br />
<strong>Doing another PCR round of α (SAMS peptide): </strong> <br> <br />
<strong>α: PCR fusion of cfp-SAMS and yfp: </strong>Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br> <br />
<br> <br />
α1: regular protocol. <br> <br />
α2: regular protocol. <br> <br />
α3: 1.25 µl extra primer 9.<br> <br />
α4: 1.25 µl extra primer 12. <br> <br />
α5: 1.25 µl extra primer 9 and 1.25 µl primer 12. <br> <br />
αK: Control<br> <br />
Regular PCR-settings</p></li> <br />
<li><p> <strong>2010-07-19</strong><br> <br />
Karl, Peidi and Kemal<br> <br />
<br> <br />
<strong>Doing a new experimental setup for IV: </strong><br> <br />
<strong>IV. PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br> <br />
<br> <br />
13 different tubes were prepared and run at two different PCR-settings. Block A: Regular settings except double annealing time (1 min). Block B: Regular settings except 58° C annealing temperature. <br> <br />
<br> <br />
C : Control<br> <br />
1a: same as 2* (template 5 5 x diluted) <br> <br />
1b. same as 3* (template 5 10 x diluted) <br> <br />
1c: same as 4* (template 5 100x diluted) <br> <br />
2a: increased product A concentration to 5 ng/µl. <br> <br />
2b: increased product A concentration to 10 ng/µl. <br> <br />
3a: same as 5* (2x primer 7) <br> <br />
3b: same as 6* (template 5 diluted 5x, 2x primer 7) <br> <br />
3c: same as 7* (template 5 diluted 10x, 2x primer 7) <br> <br />
3d same as 8* (template 5 diluted 100x, 2x primer 7) <br> <br />
4a: 2x primer 7. <br> <br />
4b: 2x primer 7, 5x template A. <br> <br />
4c: 2x primer 7, 10 x template A. <br></p></li> <br />
<li><p> <strong>2010-07-16</strong><br> <br />
Lokesh, Kemal, Katarina and Peidi<br> <br />
<br> <br />
<strong>Verification of PCR </strong>reactions IV* (8 tubes) and α on a 0.7 % agarose gel (5 µl sample+ 2µl loading buffer, GeneRuler 1 kb DNA ladder). <br> <br />
<br> <br />
No bands visible. None of the PCR reactions worked, just small bands visible corresponding to primers. The α PCR reaction messed the tubes up. That might be an explanation why there were no bands on the gel for α. A new experimental setup must be made tomorrow. <br> <br />
<br> <br />
PCR products I and 2 were run with a low range ladder to see that those products are ok. That gel was satisfactory. <br> <br />
<br></p> <br />
<li><p><strong>2010-07-15</strong><br> <br />
Malin, Julia, Kemal and Karl<br> <br />
<br> <br />
<strong>Concentrations of PCR products: </strong><br> <br />
4.2. 356.2 ng/µl<br> <br />
I. 338.3 ng/µl<br> <br />
II. 303.6 ng/µl<br> <br />
III. 337.5 ng/µl<br> <br />
IV. 280.9 ng/µl<br> <br />
<br> <br />
All PCR-products were diluted to 1 ng/µl. <br> <br />
<br> <br />
<strong>Verified PCR products</strong> I-IV and 4.2 on a 0.7 % Agarose Gel (8 µl sample + 2µl loading buffer, GeneRuler 1 kb DNA ladder) <br> <br />
PCR products I, II and III were good but 4.2 showed no bands at all. After primer design control we found a problem with primer 14, which then was ordered again. IV had only a band around 700 bp instead of 2600. This might be template 5, which then has been amplified. If the settings are changed maybe it will work. Firstly the template 5 will be diluted 0x, 5x, 10x and 100x (4 tubes: 1*, 2*, 3* and 4*) and these dilutions will also be run with higher primer concentration 7 (4 tubes: 5*, 6*, 7* and 8*). 2.5 µl primer 7 will be added instead of 1.25 µl primer 7. <br> <br />
<br> <br />
New PCR reactions were run: <br> <br />
<strong>α: PCR fusion of cfp-SAMS and yfp: </strong> Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br> <br />
K4. Control. <br> <br />
<br> <br />
<strong>IV *: PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br> <br />
Half volume 25 µl. Regular PCR settings except 56 °C annealing temperature. <br> <br />
1*;- 8*<br> <br />
K1*. Control for 1-4*.<br> <br />
K2*. Control for 5-8*.<br> <br />
<br></p></li> <br />
<br />
<li> <p><strong>2010-07-14</strong><br> <br />
Julia and Katarina <br> <br />
<strong>Ran PCR 1-5.</strong><br> <br />
1. Amplification of cfp-sams compatible: Add restriction enzyme site BamHI on N-terminus +SAMS on C-terminus.<br> <br />
Expected fragment length: 748 bp.<br> <br />
2. Amplification of yfp-SAMS competible: Adds restriction enzyme site for HindIII on C-terminus and SAMS tail on N-terminus. <br> <br />
Expected fragment length: 762 bp.<br> <br />
3. Amplification of yfp-snf4 compatible: Adds restriction enzyme site BamHI on N-terminus and Snf4 tail on C-terminus. <br> <br />
Expected fragment length: 755 bp.<br> <br />
4. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. <br> <br />
Expected fragment length: 769 bp.<br> <br />
5. Amplification of cfp-snf1 compatible: Adds restriction enzyme site SpeI on N-terminus and Snf1 tail on C-terminus.<br> <br />
Expected fragment length: 757 bp.<br> <br />
K2. Control K2.<br> <br />
<br> <br />
Julia, Katarina and Malin<br> <br />
<strong>Verified PCR 1-5:</strong> measured concentration by nanodrop and checked if the fragments were the correct length by running on a 2 % agarose gel (8 µl sample+2 µl loading buffer, Fermentas FastRuler Low Range).<br> <br />
PCR product 1, 2, 3 and 5 worked and gave expected fragment lengths. Number 4 however did not work. Only a fragment around 80 bp was visible, probably a primer dimer.<br> <br />
<br> <br />
<strong>Concentrations:</strong><br> <br />
1. 443.9 ng/µl<br> <br />
2. 371.0 ng/µl<br> <br />
3. 361.7 ng/µl<br> <br />
4. --<br> <br />
5. 377.6 ng/µl<br> <br />
A. 451.1 ng/µl (snf1)<br> <br />
B. 438.7 ng/µl (snf4)<br> <br />
C. 408.9 ng/µl (SAMS) <br> <br />
1-5 and A-C were diluted to 1 ng/µl.<br> <br />
<br> <br />
<strong>Ran PCR of I, II, III, IV and 4.</strong><br> <br />
4.2. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. Expected fragment length: 769 bp. (second try)<br> <br />
I. PCR fusion of cfp and SAMS: Adds BamHI on N-terminus of cfp and gives a SAMS C-terminus. Expected fragment length: 768 bp.<br> <br />
II. PCR fusion of yfp and Snf4 1: Adds BamHI on N-terminus of yfp and removes stop codon. Expected fragment length: 1694 bp.<br> <br />
III. PCR fusion of yfp and Snf4 2: Adds BamHI on N-terminus of yfp and adds HindIII on C-terminus of Snf4 plus adds stop codon. Expected fragment length: 1708 bp. Final Product!<br> <br />
IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.<br> <br />
K3. Control<br> <br />
<br> <br />
Karl and Malin<br> <br />
Transferred diploid cells of snf1Δ mutants to two sporulation plates and incubated in 30° C. (For 2-7 days)<br> <br />
</p></li> <br />
<br />
<li><p><strong>2010-07-13</strong><br> <br />
Julia and Lokesh<br> <br />
Ran the gel with products of our first three PCRs along with the templates we made for the two fluorescent proteins. We also prepared the sample solutions of DNA templates with appropriate concentration (1ng/µl) for the next PCR reactions.<br> <br />
<strong>Verified</strong> the PCR products and the digested miniprepped EYFP and ECFP by running an agarose gel which showed good results. Expected fragment lengths were received. The SAMS-peptide however was too short to be visible on the gel (67 bp).<br> <br />
<strong>Prepared for PCR 1-5. </strong>The miniprepped ECFP and EYFP were diluted to a stock concentration of 1 ng/ ml to use for the PCR. The concentrations of the first PCR product were measured using nanodrop and then diluted to 1 ng/ml.<br> <br />
snf1: 20.5 ng/ml<br> <br />
snf4: 122.6 ng/ml<br> <br />
SAMS: 114.9 ng/ml<br> <br />
</p> <br />
<p> Karl and Adnan<br> <br />
We made duplicates of the haploid Snf4∆ and wildtype strains on new plates in case the original plate would be contaminated. Prepared a sporulation medium that was autoclaved and poured into empty growth plates to stay in room temperature over night.</p> <br />
<br></li> <br />
<li> <p> <strong>2010-07-12</strong><br> <br />
Katarina and Julia<br> <br />
<strong>Amplification of genomic SNF1 DNA by PCR. </strong>All primers were resuspended in mq-water according to the volume on the lable, the final concentration 100 pg/ml. The primers were then diluted 10x and the SAMS-peptide templates were diluted 1000x. PCR reactions were run amplifying subunits Snf1 and Snf4 and also the SAMS-peptide. YPD medium and agar plates were prepared (yeast).<br> <br />
</p> <br />
<p> Adnan and Kemal<br> <br />
<strong>Miniprep purification </strong>of EYFP and ECFP plasmides from overnight culture. The concentrations were measured using Nanodrop:<br> <br />
ECFP1: 99 ng/ml<br> <br />
ECFP2: 89.9<br> <br />
EYFP1: 104, 7 <br> <br />
EYFP2: 172,5<br> <br />
The concentrations were verified with electrophoresis. The plasmids were cut with restriction enzymes EcoRI and Pst1.<br> <br />
ECFP, plasmid pSB1AK3, fragment sizes 750 bp and 3200 bp.<br> <br />
EYFP, plasmid pSB1A2, fragment sizes 750 bp and 2050 bp.</p></li> <br />
<br> <br />
<li><p><strong>2010-07-11</strong><br> <br />
Katarina<br> <br />
Made overnight cultures with colony 3 and 4 plasmid (not single cultures) and twp each of EYFP and ECFP.<br />
</p> <br />
</li> <br />
<br> <br />
<li><p><strong>2010-07-10</strong><br> <br />
Julia<br> <br />
Moved plated bacteria from incubator to refrigerator. <br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-09</strong><br> <br />
Malin and Julia<br> <br />
The SOC medium was autoclaved. Competent E.coli cells were transformed with our FPs through heat shock and plated on petri dishes. 20 new LB-amp plates were made.<br> <br />
<br />
Adnan and Katarina<br> <br />
Picked up primers at Chalmers and checked if they were ok. The concentration of the genomic DNA (SNF1 gene) were measured. Prepared a master mix so that the PCR-reactions could be started Monday morning.</p> <br />
<br> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-08</strong><br> <br />
Malin, Julia and Peidi<br> <br />
The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-07 </strong><br> <br />
Adnan, Katarina and Julia<br> <br />
As the gels from Tuesday showed ambigous results we decided to redo the miniprep of the plamids. We used the overnight preparations of the two unused colonies (3 &amp; 4). The miniprep went smoothly except for major difficulties regarding untrustworthy pipettes. The pipettes marked with green tape are the ones to use from now on together with the tips with the same markings! A gel was molded and this time we decided to use a full gel to get a better resolution. The results on the gel were very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-06</strong><br> <br />
Malin, Adnan and Peidi<br> <br />
Colony 1 and 2 were picked to do a miniprep. The plasmids were extracted and a plasmid integrity check was performed. By cutting with BamHI, one cut were made to get the size of the plasmid. Then we cut with ClaI which is a two-cutter that gives a fragment that is 600 bp and one that is 6800. The gel which should confirm this was not satisfactory since the 600 band was missing.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-05</strong><br> <br />
Malin and Katarina<br> <br />
An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1.</p> <br />
</li> <br />
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<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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http://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note
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<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/f/fe/Gfp.png" width="40" height="40" align="left"></div> <strong><p>Fluorescent Proteins:</strong><br> Green Fluorescent Protein <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p><br> <br />
<br />
<div class="logo"> <img src=" https://static.igem.org/mediawiki/2010/a/a9/Plasmid.png" width="40" height="40" align="left"></div><p><strong>Plasmid backbone:</strong> <br><br />
pSP-GM1 <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#plasmid">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/7/74/Snf1Com.png" width="40" height="40" align="left"></div><p> <strong>SNF1 (modified subunits):</strong><br><br />
Snf1 (alfa), Snf4 (gamma) <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#SNF1">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/4/46/FpPos.png" width="40" height="40" align="left"></div> <p> <strong>FP positions:</strong> <br><br />
N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#position">(read more)</a></p> <br> <br />
<br />
<div class="logo"> <img src="https://static.igem.org/mediawiki/2010/e/e0/PrDesign.png" width="40" height="40" align="left"></div> <p> <strong>Primer design:</strong><br> <br />
Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#primer">(read more)</a></p> <br> <br />
<br />
<br />
<br />
<div id="myTitle">Lab work by date </div> <br />
<ul><li><p> <strong>2010-08-11</strong><br> <br />
Malin and Katarina<br> <br />
<br> <br />
The PCR reaction from yesterday was checked on a gel. α should be around 1500 bp but only a band around 750 bp was seen. We had a long meeting with supervisor Per about PCR reactions and the next couple of weeks in the lab. <br> <br />
<br> <br />
Karl and Kemal <br> <br />
<br> <br />
Ran PCR reaction α with different settings. <br> <br />
<strong>α1: </strong>regular settings<br> <br />
<strong>α2: </strong>10 x diluted template 2<br> <br />
<strong>α3: </strong>increased annealing time from 30 s to 60 s<br> <br />
<strong>α4: </strong>decreased annealing temperature<br> <br />
<br> <br />
After PCR, the products were run on a gel. α1, α3 and α 4 had a band around 750 bp but nothing on α2. </p></li><br> <br />
<br> <br />
<li><p> <strong>2010-08-10</strong><br> <br />
Peidi and Lokesh<br> <br />
<br> <br />
Checked the transformation plates but nothing had grown. Prepared PCR reaction Γ. <br> <br />
<br> <br />
Katarina and Kemal<br> <br />
<br> <br />
PCR product Γ was verified on a 0.7 % agarose gel. There was a band around 750 bp as expected and it was purified using the regular protocol. A new phusion mastermix was made and PCR reaction α was run with Γ as one of the template. Regular phusion PCR settings was used. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-08-09</strong><br> <br />
Malin and Kemal<br> <br />
<br> <br />
Before checking if the ligation worked we need to transform and amplify in E.coli. Then we can miniprep the ligated plasmid and check on a gel. After that we can digest plasmid with PacI and BcuI and ligate the digested IV. Then another round of transformation and miniprep before we can transform yeast. <br> <br />
<br> <br />
Karl and Lokesh<br> <br />
<br> <br />
Transformed competent E.coli with plasmid ligated with III by heat shock. The cells were incubated at 37 °C over night. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-07</strong><br> <br />
Lokesh<br> <br />
<br> <br />
Moved ligation tube to freezer. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-06</strong><br> <br />
Malin, Julia and Lokesh<br> <br />
<br> <br />
III and IV were digested 3x protocol and were then put on a 1 % agarose gel together with Γ both for verification and purification. Correct bands were seen for purification except for Γ which was empty. In the verification lanes the digested PCR products were to light to be seen but they were seen in purification lanes. IIId and IVd were purified. <br> <br />
<br> <br />
<strong>IIId: </strong>7.8 ng/µl<br> <br />
<strong>IVd: </strong>11.7ng/µl<br> <br />
<br> <br />
IIId will now be ligated with the cut plasmid (BamHI and HindIII). Ligation performed according to protocol with 156 ng insert and 52 ng plasmid. Ligation was incubated over night in fridge at 4-16° C. <br> <br />
<br> <br />
All 4 sporulation plates were checked but still no tetrads visible. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-05</strong><br> <br />
Malin and Julia<br> <br />
<br> <br />
III and IV were digested using the regular protocol. <br> <br />
<strong>III: </strong>HindIII and BamHI works with Buffer B<br> <br />
<strong>IV: </strong>PacI and BcuI works with Buffer M<br> <br />
<br> <br />
A 1% gel was run with uncut and digested III and IV, control, I and Β. I worked well and will be purified. The Β lane showed no bands. None of the digested products were visible on the gel so a new gel was run trying to verify this result. The control shows a band around 1700 which is weird. This band is also visible on the III uncut but together with a band around 750 bp. No bands at the IV which is very strange since this PCR product has been verified before. Have something gone wrong with the purification? <br> <br />
<br> <br />
As a result PCR reactions III and IV will be redone. New PCR reactions of Γ and α were also performed in the same block. <br> <br />
<br> <br />
Karl and Kemal<br> <br />
<br> <br />
Reactions of Β were made. Β1 is a continuation of the 100 cycle PCR this time with primers. Β2 uses a megaprimer approach. <br> <br />
<br> <br />
All the PCR products were run on a 1 % agarose gel. III, IV and Γ were alright. α and Β2 showed a band around 750 bp. <br> <br />
<strong>III: </strong>531.3 ng/µl<br> <br />
<strong>IV: </strong>263.9 ng/µl<br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-04</strong><br> <br />
Julia and Karl<br> <br />
<br> <br />
PCR products Β were loaded on a 0.7 % gel. No correct bands, only amplified templates around 750 bp and something else around 500 bp. <br> <br />
<br> <br />
PCR of SAMS will also be redone from scratch. PCR reactions 1, 2 and C are done, then loaded on a gel (2 % gel and low range ladder) and purified. <br> <br />
<strong>1: </strong>8.2 ng/µl<br> <br />
<strong>2: </strong>7.8 ng/µl<br> <br />
<strong>C: </strong>4.9 ng/µl<br> <br />
Samples diluted to 1 ng/µl. <br> <br />
<br> <br />
PCR of Β was done again but this time in two steps and with the Finnzymes Tm which corresponds to an annealing temp at 85 °C. Firstly 10 cycles without primers were performed. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
Second round of PCR with primers were performed. Then new PCR reactions amplifying III and IV were done and also reaction I. <br> <br />
<strong>III: </strong>572 ng/µl<br> <br />
<strong>IV: </strong>660 ng/µl<br> <br />
<br> <br />
The sporplates were checked and very few tetrads were seen. <br> <br />
<br> <br />
Β samples were put on a gel but no correct bands visible. We suspect that the reverse primer is attaching in the wrong place since YFP and CFP is so similar which then would lead to 750 bp fragment. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-03</strong><br> <br />
Malin and Lokesh<br> <br />
<br> <br />
PCR products II, III and IV were verified and purified from a 1 % agarose gel. III did not work and was not purified. <br> <br />
<br> <br />
Concentrations: <br> <br />
<strong>II: </strong>6.2 ng/µl <br> <br />
<strong>IV: </strong>5.6 ng/µl<br> <br />
II and IV were diluted to 1 ng/µl. <br> <br />
<br> <br />
The new sporplates were examined but no tetrads visible. To check further the cells were stained with DAPI and examined in fluorescence microscope. Still no success. <br> <br />
<br> <br />
New PCR reactions were prepared. III was redone and Β was also done. Same settings as yesterday. <br> <br />
<br> <br />
Kemal and Karl<br> <br />
<br> <br />
PCR products were loaded on a 1% agarose gel for verification and purification. III worked this time and was purified from the gel using the protocol from QIAquick Gel Extraction Kit. <br> <br />
<strong>III: </strong>6.4 ng/µl<br> <br />
<br> <br />
New PCR of Β was run with two different block settings. <br> <br />
<strong>Block A: </strong>increased annealing time to 1 min. <br> <br />
<strong>Block B: </strong>increased annealing time to 58 °C. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-08-02</strong><br> <br />
Kemal, Lokesh and Karl<br> <br />
<br> <br />
PCR products A, B and 3 were loaded on a gel and then purified from the gel using the protocol from QIAquick Gel Extraction Kit. α and IV were also run on a gel but only showed strong bands corresponding to amplified templates. On the α lane however a very light band was seen at the correct length. This will be further investigated. <br> <br />
<br> <br />
Malin and Julia<br> <br />
<br> <br />
The purified PCR products were measured with nanodrop: <br> <br />
<strong>A1: </strong>11.8 ng/µl<br> <br />
<strong>A2: </strong>10.2 ng/µl<br> <br />
<strong>B1: </strong>9.6 ng/µl<br> <br />
<strong>B2: </strong>17.1 ng/µl<br> <br />
<strong>31: </strong>15.0 ng/µl<br> <br />
<strong>32: </strong>16.3 ng/µl<br> <br />
<br> <br />
A, B and 3 were diluted to 1 ng/µl. <br> <br />
<br> <br />
PCR products 4 and 5 were loaded on a 1 % agarose gel together with IV that should be controlled. PCR products 4 and 5 were correct so they were purified from the gel using the protocol from QIAquick Gel Extraction Kit. IV however was not correct. Only a band around 750 bp was visible. <br> <br />
<br> <br />
Concentrations: <br> <br />
<strong>4: </strong>10.8 ng/µl<br> <br />
<strong>5: </strong>10.4 ng/µl<br> <br />
4-5 were diluted to 1 ng/µl. <br> <br />
New PCR reactions were started (II, III and IV). PCR settings that works well with the Phusion enzyme was set: <br> <br />
Initial denaturation: 98 °C 3 min<br> <br />
Denaturation: 98 °C 10 s<br> <br />
Annealing: 55 °C 30 s<br> <br />
Elongation: 72 °C 1.5 min+ 1s/cycle<br> <br />
30 cycles<br> <br />
Final extension: 72 °C 10 min<br> <br />
4°C forever <br> <br />
<br> <br />
<strong>2010-07-30</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
The concentration of the purified plasmids were measured with nanodrop: <br> <br />
<strong>P1: </strong>1.7 ng/µl<br> <br />
<strong>P2: </strong>3.5 ng/µl<br> <br />
<strong>P3: </strong>25.9 ng/µl<br> <br />
<strong>P4: </strong>42.8 ng/µl<br> <br />
This is clear evidence that water should be used for eluation in the future! <br> <br />
<br> <br />
To make sure that we have the right products after purification a 1 % agarose gel was run with fastruler DNA ladder. The gel verified the plasmid size though the bands were light. <br> <br />
<br> <br />
PCR product III was evaporated since it was so dilute. New concentration: <br> <br />
<strong>IIId: </strong>35 ng/µL<br> <br />
<br> <br />
The digested PCR product III and the digested plasmid were ligated with T4 ligase. The DNA concentration in total should not exceed 1 µg. The ratio insert:vector should be 3:1. <br> <br />
<br> <br />
<strong>Ligation protocol: </strong><br> <br />
150 ng insert<br> <br />
50 ng vector<br> <br />
3 µl 10x T4 buffer<br> <br />
2.5 µl (2.5 U, between 1-5 U) T4 ligase<br> <br />
Adjust volume with water up to 30 µl. Mix and incubate in 4-16 °C (refrigerator) overnight. <br> <br />
The newly plated sporplates were moved to 30°C. The old sporplates were again studied in the microscope and no tetrads were seen. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
Since we have had a lot of trouble with the longer fusions we will start over from scratch with a new high fidelity polymerase called “Finnzumes Phusion”. New PCR reactions were run (A, B, 3, 4 and 5). IV and α were also performed using alternative settings (2 different PCR reactions described before) but with “old” templates. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-29</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
PCR product III is the final fusion protein where Snf4 is tagged with yfp. To be able to insert the construct into the plasmid we must digest it. <br> <br />
<br> <br />
Digestion: Mix DNA, buffer and water. Last add enzyme and mix. Incubate at 37° C for 1 hour. <br> <br />
1 µg DNA<br> <br />
2.5 µl Buffer<br> <br />
1 U Enzyme<br> <br />
Up to 25 µl H2O<br> <br />
For PCR product III, the restriction enzymes BamHI and HindIII are used together with buffer B. After digestion (the protocol was scaled 3x) the sample was loaded on a 1 % agarose gel in 6 lanes with 1 kb ladder. The bands were cut out and weighed. The samples were purified from the gel using the protocol from QIAquick Gel Extraction Kit. The DNA was eluated with water. <br> <br />
<br> <br />
Concentration: <br> <br />
<strong>IIId: </strong>5 ng/µl (Total product 160 µl= 800 ng) <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
The plasmids were also digested with BamHI and HindIII according to the same protocol as above. P1 and P2 were eluated with buffer EB and P3 and P4 with water. <br> <br />
<br> <br />
2 new sporulation plates were prepared by plating the diploid yeast cells on spore plates and kept at room temperature. <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-28</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
The PCR products were run on a 1 % gel with 1 kb ladder. Γ may have worked. α and Β had a band around 750 bp and a weird light band around 500 bp. They obviously did not work. IV did not work at all. <br> <br />
<br> <br />
The sporulation plates were checked for spores. Cells were studied under a microscope. No tetrads visible. <br> <br />
<br></p></li> <br />
<li><p> <strong>2010-07-27</strong><br> <br />
Julia and Katarina<br> <br />
<br> <br />
A 1.5 % gel was run with all of the fragments around 700 bp to control the validity of the PCR reactions. A low range ladder was used. PCR products 1-5 seems good. The results from yesterday were confirmed. <br> <br />
<br> <br />
A new PCR design was made with α, Β, Γ and IV. PCR was done in two steps to increase the chance of success for longer fusions. <br> <br />
1. 7 cycles with short annealing time. <br> <br />
2. 20 cycles with long annealing time (all other settings kept standard). <br> <br />
<br></p></li> <br />
<li><p><strong>2010-07-26</strong><br> <br />
<br> <br />
A new experiment were designed called Γ which is the same as I but with longer annealing region. Also an experiment called Δ were designed which corresponds to II. <br> <br />
<br> <br />
Julia and Katarina<br> <br />
<br> <br />
PCR reactions were run with samples of 4, IV and Γ. IV was tried with two different PCR settings: one regular and one with two runs of PCR, the first without primers and only 5 cycles and the second one with regular settings. <br> <br />
<br> <br />
Kemal and Lokesh<br> <br />
<br> <br />
The PCR products were controlled on a 0.7% agarose gel. Only 4 worked. Γ was empty and IV had bands in the wrong place (amplified template). <br> <br />
<strong>4: </strong>534 ng/ µl </p></li> <br />
<li><p> <strong>2010-07-22</strong><br> <br />
Karl, Lokesh and Kemal<br />
</br></br> <br />
A new mastermix was prepared using a different enzyme. Then the experiments from yesterday was redone using regular PCR settings: <br> <br />
A, 5, 2, I<br> <br />
Then some more experiments of IV was redone: <br> <br />
2a, 2b and 2c<br> <br />
The new primer 14 had arrived so PCR reaction 4 was also redone. <br> <br />
<br> <br />
All the PCR products were run on a 0.7 % agarose gel with GeneRuler 1 kb DNA ladder. The results were inconclusive. None of the expected bands were received. At this point we are not sure what to do since none of the PCR reactions worked. <br> <br />
<br> <br />
</p> <br />
</li> <br />
<li> <p> <strong>2010-07-21</strong><br> <br />
Malin, Julia, Karl and Lokesh<br> <br />
<br> <br />
<strong>Verification of the PCR products </strong>α1, α2, α3, α4, α5 and αK by running on a thin 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder). <br> <br />
<br> <br />
The gel didn’t show the expected band lengths. A band around 700 bp could be seen, most strongly for α4 and α5. We suspect that there is something wrong with the ladder because we get inconclusive results. Since none of the PCR reactions for the last couple of days have worked we will redo the templates of α and IV: I, 2, 5 and A. These PCR reactions have worked before. <br> <br />
<br> <br />
<strong>Expected fragment length: </strong><br> <br />
A.2: 1938 bp<br> <br />
5.2: 757 bp<br> <br />
2.2: 762 bp<br> <br />
I.2: 768 bp<br> <br />
<br> <br />
The PCR products were run on a thin 0.7 % agarose gel. We used both the FastRuler High Range Ladder and the GeneRuler 1 kb DNA ladder. 5.2 and 2.2 showed the expected bands around 750 bp. I.2 and A.2 did not work at all, nothing visible on the gel. We also run some of the old PCR products to see what the result would be using a new ladder. We run α2, α5, 4c (IV) and 4c*(IV). Here some template amplifications were visible but sadly none of the expected fragment lengths. Both ladders showed the same result so there is probably nothing wrong with the GeneRuler ladder except that it is not showing strong color. <br></p></li> <br />
<li><p><strong>2010-07-20</strong><br> <br />
Malin, Julia, Lokesh and Karl<br> <br />
<br> <br />
<strong>Verification of yesterdays PCR reactions </strong>by running on a 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder) <br> <br />
<br> <br />
None of the reactions worked. <br> <br />
<br> <br />
Gel 1 (Block A): Nothing visible on gel, ladders only very light so these samples were run on a new 0.7 % agarose gel but this time thinner to get better resulotion (0.58 g agarose + 80 ml TBE). This time no expected fragments were seen, but bands corresponding to primer dimers were clearly seen. <br> <br />
Gel 2 (Block B): A few small bands were seen but no bands of expected length. <br> <br />
<br> <br />
We decided to stop working on IV until proper feedback on the experimental setup was received. Instead we continued with α.<br> <br />
<br> <br />
<strong>Doing another PCR round of α (SAMS peptide): </strong> <br> <br />
<strong>α: PCR fusion of cfp-SAMS and yfp: </strong>Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br> <br />
<br> <br />
α1: regular protocol. <br> <br />
α2: regular protocol. <br> <br />
α3: 1.25 µl extra primer 9.<br> <br />
α4: 1.25 µl extra primer 12. <br> <br />
α5: 1.25 µl extra primer 9 and 1.25 µl primer 12. <br> <br />
αK: Control<br> <br />
Regular PCR-settings</p></li> <br />
<li><p> <strong>2010-07-19</strong><br> <br />
Karl, Peidi and Kemal<br> <br />
<br> <br />
<strong>Doing a new experimental setup for IV: </strong><br> <br />
<strong>IV. PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br> <br />
<br> <br />
13 different tubes were prepared and run at two different PCR-settings. Block A: Regular settings except double annealing time (1 min). Block B: Regular settings except 58° C annealing temperature. <br> <br />
<br> <br />
C : Control<br> <br />
1a: same as 2* (template 5 5 x diluted) <br> <br />
1b. same as 3* (template 5 10 x diluted) <br> <br />
1c: same as 4* (template 5 100x diluted) <br> <br />
2a: increased product A concentration to 5 ng/µl. <br> <br />
2b: increased product A concentration to 10 ng/µl. <br> <br />
3a: same as 5* (2x primer 7) <br> <br />
3b: same as 6* (template 5 diluted 5x, 2x primer 7) <br> <br />
3c: same as 7* (template 5 diluted 10x, 2x primer 7) <br> <br />
3d same as 8* (template 5 diluted 100x, 2x primer 7) <br> <br />
4a: 2x primer 7. <br> <br />
4b: 2x primer 7, 5x template A. <br> <br />
4c: 2x primer 7, 10 x template A. <br></p></li> <br />
<li><p> <strong>2010-07-16</strong><br> <br />
Lokesh, Kemal, Katarina and Peidi<br> <br />
<br> <br />
<strong>Verification of PCR </strong>reactions IV* (8 tubes) and α on a 0.7 % agarose gel (5 µl sample+ 2µl loading buffer, GeneRuler 1 kb DNA ladder). <br> <br />
<br> <br />
No bands visible. None of the PCR reactions worked, just small bands visible corresponding to primers. The α PCR reaction messed the tubes up. That might be an explanation why there were no bands on the gel for α. A new experimental setup must be made tomorrow. <br> <br />
<br> <br />
PCR products I and 2 were run with a low range ladder to see that those products are ok. That gel was satisfactory. <br> <br />
<br></p> <br />
<li><p><strong>2010-07-15</strong><br> <br />
Malin, Julia, Kemal and Karl<br> <br />
<br> <br />
<strong>Concentrations of PCR products: </strong><br> <br />
4.2. 356.2 ng/µl<br> <br />
I. 338.3 ng/µl<br> <br />
II. 303.6 ng/µl<br> <br />
III. 337.5 ng/µl<br> <br />
IV. 280.9 ng/µl<br> <br />
<br> <br />
All PCR-products were diluted to 1 ng/µl. <br> <br />
<br> <br />
<strong>Verified PCR products</strong> I-IV and 4.2 on a 0.7 % Agarose Gel (8 µl sample + 2µl loading buffer, GeneRuler 1 kb DNA ladder) <br> <br />
PCR products I, II and III were good but 4.2 showed no bands at all. After primer design control we found a problem with primer 14, which then was ordered again. IV had only a band around 700 bp instead of 2600. This might be template 5, which then has been amplified. If the settings are changed maybe it will work. Firstly the template 5 will be diluted 0x, 5x, 10x and 100x (4 tubes: 1*, 2*, 3* and 4*) and these dilutions will also be run with higher primer concentration 7 (4 tubes: 5*, 6*, 7* and 8*). 2.5 µl primer 7 will be added instead of 1.25 µl primer 7. <br> <br />
<br> <br />
New PCR reactions were run: <br> <br />
<strong>α: PCR fusion of cfp-SAMS and yfp: </strong> Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br> <br />
K4. Control. <br> <br />
<br> <br />
<strong>IV *: PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br> <br />
Half volume 25 µl. Regular PCR settings except 56 °C annealing temperature. <br> <br />
1*;- 8*<br> <br />
K1*. Control for 1-4*.<br> <br />
K2*. Control for 5-8*.<br> <br />
<br></p></li> <br />
<br />
<li> <p><strong>2010-07-14</strong><br> <br />
Julia and Katarina <br> <br />
<strong>Ran PCR 1-5.</strong><br> <br />
1. Amplification of cfp-sams compatible: Add restriction enzyme site BamHI on N-terminus +SAMS on C-terminus.<br> <br />
Expected fragment length: 748 bp.<br> <br />
2. Amplification of yfp-SAMS competible: Adds restriction enzyme site for HindIII on C-terminus and SAMS tail on N-terminus. <br> <br />
Expected fragment length: 762 bp.<br> <br />
3. Amplification of yfp-snf4 compatible: Adds restriction enzyme site BamHI on N-terminus and Snf4 tail on C-terminus. <br> <br />
Expected fragment length: 755 bp.<br> <br />
4. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. <br> <br />
Expected fragment length: 769 bp.<br> <br />
5. Amplification of cfp-snf1 compatible: Adds restriction enzyme site SpeI on N-terminus and Snf1 tail on C-terminus.<br> <br />
Expected fragment length: 757 bp.<br> <br />
K2. Control K2.<br> <br />
<br> <br />
Julia, Katarina and Malin<br> <br />
<strong>Verified PCR 1-5:</strong> measured concentration by nanodrop and checked if the fragments were the correct length by running on a 2 % agarose gel (8 µl sample+2 µl loading buffer, Fermentas FastRuler Low Range).<br> <br />
PCR product 1, 2, 3 and 5 worked and gave expected fragment lengths. Number 4 however did not work. Only a fragment around 80 bp was visible, probably a primer dimer.<br> <br />
<br> <br />
<strong>Concentrations:</strong><br> <br />
1. 443.9 ng/µl<br> <br />
2. 371.0 ng/µl<br> <br />
3. 361.7 ng/µl<br> <br />
4. --<br> <br />
5. 377.6 ng/µl<br> <br />
A. 451.1 ng/µl (snf1)<br> <br />
B. 438.7 ng/µl (snf4)<br> <br />
C. 408.9 ng/µl (SAMS) <br> <br />
1-5 and A-C were diluted to 1 ng/µl.<br> <br />
<br> <br />
<strong>Ran PCR of I, II, III, IV and 4.</strong><br> <br />
4.2. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. Expected fragment length: 769 bp. (second try)<br> <br />
I. PCR fusion of cfp and SAMS: Adds BamHI on N-terminus of cfp and gives a SAMS C-terminus. Expected fragment length: 768 bp.<br> <br />
II. PCR fusion of yfp and Snf4 1: Adds BamHI on N-terminus of yfp and removes stop codon. Expected fragment length: 1694 bp.<br> <br />
III. PCR fusion of yfp and Snf4 2: Adds BamHI on N-terminus of yfp and adds HindIII on C-terminus of Snf4 plus adds stop codon. Expected fragment length: 1708 bp. Final Product!<br> <br />
IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.<br> <br />
K3. Control<br> <br />
<br> <br />
Karl and Malin<br> <br />
Transferred diploid cells of snf1Δ mutants to two sporulation plates and incubated in 30° C. (For 2-7 days)<br> <br />
</p></li> <br />
<br />
<li><p><strong>2010-07-13</strong><br> <br />
Julia and Lokesh<br> <br />
Ran the gel with products of our first three PCRs along with the templates we made for the two fluorescent proteins. We also prepared the sample solutions of DNA templates with appropriate concentration (1ng/µl) for the next PCR reactions.<br> <br />
<strong>Verified</strong> the PCR products and the digested miniprepped EYFP and ECFP by running an agarose gel which showed good results. Expected fragment lengths were received. The SAMS-peptide however was too short to be visible on the gel (67 bp).<br> <br />
<strong>Prepared for PCR 1-5. </strong>The miniprepped ECFP and EYFP were diluted to a stock concentration of 1 ng/ ml to use for the PCR. The concentrations of the first PCR product were measured using nanodrop and then diluted to 1 ng/ml.<br> <br />
snf1: 20.5 ng/ml<br> <br />
snf4: 122.6 ng/ml<br> <br />
SAMS: 114.9 ng/ml<br> <br />
</p> <br />
<p> Karl and Adnan<br> <br />
We made duplicates of the haploid Snf4∆ and wildtype strains on new plates in case the original plate would be contaminated. Prepared a sporulation medium that was autoclaved and poured into empty growth plates to stay in room temperature over night.</p> <br />
<br></li> <br />
<li> <p> <strong>2010-07-12</strong><br> <br />
Katarina and Julia<br> <br />
<strong>Amplification of genomic SNF1 DNA by PCR. </strong>All primers were resuspended in mq-water according to the volume on the lable, the final concentration 100 pg/ml. The primers were then diluted 10x and the SAMS-peptide templates were diluted 1000x. PCR reactions were run amplifying subunits Snf1 and Snf4 and also the SAMS-peptide. YPD medium and agar plates were prepared (yeast).<br> <br />
</p> <br />
<p> Adnan and Kemal<br> <br />
<strong>Miniprep purification </strong>of EYFP and ECFP plasmides from overnight culture. The concentrations were measured using Nanodrop:<br> <br />
ECFP1: 99 ng/ml<br> <br />
ECFP2: 89.9<br> <br />
EYFP1: 104, 7 <br> <br />
EYFP2: 172,5<br> <br />
The concentrations were verified with electrophoresis. The plasmids were cut with restriction enzymes EcoRI and Pst1.<br> <br />
ECFP, plasmid pSB1AK3, fragment sizes 750 bp and 3200 bp.<br> <br />
EYFP, plasmid pSB1A2, fragment sizes 750 bp and 2050 bp.</p></li> <br />
<br> <br />
<li><p><strong>2010-07-11</strong><br> <br />
Katarina<br> <br />
Made overnight cultures with colony 3 and 4 plasmid (not single cultures) and twp each of EYFP and ECFP.<br />
</p> <br />
</li> <br />
<br> <br />
<li><p><strong>2010-07-10</strong><br> <br />
Julia<br> <br />
Moved plated bacteria from incubator to refrigerator. <br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-09</strong><br> <br />
Malin and Julia<br> <br />
The SOC medium was autoclaved. Competent E.coli cells were transformed with our FPs through heat shock and plated on petri dishes. 20 new LB-amp plates were made.<br> <br />
<br />
Adnan and Katarina<br> <br />
Picked up primers at Chalmers and checked if they were ok. The concentration of the genomic DNA (SNF1 gene) were measured. Prepared a master mix so that the PCR-reactions could be started Monday morning.</p> <br />
<br> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-08</strong><br> <br />
Malin, Julia and Peidi<br> <br />
The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-07 </strong><br> <br />
Adnan, Katarina and Julia<br> <br />
As the gels from Tuesday showed ambigous results we decided to redo the miniprep of the plamids. We used the overnight preparations of the two unused colonies (3 &amp; 4). The miniprep went smoothly except for major difficulties regarding untrustworthy pipettes. The pipettes marked with green tape are the ones to use from now on together with the tips with the same markings! A gel was molded and this time we decided to use a full gel to get a better resolution. The results on the gel were very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-06</strong><br> <br />
Malin, Adnan and Peidi<br> <br />
Colony 1 and 2 were picked to do a miniprep. The plasmids were extracted and a plasmid integrity check was performed. By cutting with BamHI, one cut were made to get the size of the plasmid. Then we cut with ClaI which is a two-cutter that gives a fragment that is 600 bp and one that is 6800. The gel which should confirm this was not satisfactory since the 600 band was missing.<br> <br />
</p> <br />
</li> <br />
<li> <br />
<p><strong>2010-07-05</strong><br> <br />
Malin and Katarina<br> <br />
An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1.</p> <br />
</li> <br />
</ul> <br />
<br />
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</div> </div><br />
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<div class="Title3">Lab Notes</div><br />
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<div class="numleft"><br />
<p>11</p><br />
</div><br />
<div class="newsright"><a href="#">August 11, 2010</a><br />
<p> The PCR reaction from yesterday was checked on a gel. α should be ... </p><br />
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<div class="Title3">News</div><br />
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<p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...<br />
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