Team:Gothenburg-Sweden/Lab Note

From 2010.igem.org

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  <td><p class="STYLE6">Fluorescent  Proteins:</p>
  <td><p class="STYLE6">Fluorescent  Proteins:</p>
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    <p>Bioluminescence is used as a defense, offense and communication mechanism  by various organisms in nature including insects, fish, squid, sea cacti, sea  pansies, clam, shrimps and jellyfish(1). After the discovery of the  Green Fluorescent Protein (GFP) by Shimomura et al. from <em>Aequorea</em> jellyfish(2),  an unprecedented tool for visualizing living  organisms was also being introduced to molecular biology, leading to a Nobel  Prize in 2008(3). The <em>Aequorea</em> GFP is a 238 aa, 30 kDa monomer that emits green fluorescence  maximum at 509 nm wavelength(4).  It has a unique structure with an 11  stranded β - barrel like shape involving α- helixes running through the center  of the can; to which the chromophore is attached(5). Autocatalytic  formation of the chromophore of the GFP without the neccessity of any  substrates or cofactors is a rather useful property of the GFPs allowing them  to express in fusion with different proteins. Remarkably, the fusion of GFP to  a protein does not have any vital effect on the activity or mobility of the  protein in addition to its nontoxic nature(1).    </p>
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    <p>Green Fluorescent Proteins <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p>
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    <p align="center"><img src="https://static.igem.org/mediawiki/2010/7/78/Lab_note_clip_image002.jpg" alt="GFP" width="198" height="192"> <br>
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<p>&nbsp;</p>
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            </p>
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    <p align="center"><span class="STYLE7">Figure 1: Structure of the GFP taken from the  PDB, code 1EMA</span></p>
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    <p align="center">&nbsp;</p>
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    <p>References: <br>
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            </p>
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    <ul>
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      <li>(1) Zimmer, M. (2002) “Green Fluorescent Protein  (GFP): Applications, Structure and Related Photophysical Behavior.” <em>Chemical  Reviews.</em> v. 102 (3) pp. 759-781. </li>
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          <li>(2) Shimomura, O.,  Johnson, F.H. &amp; Saiga, Y. (1962) “Extraction, purification and properties  of aequorin, a bioluminescent protein from the luminous hydromedusan.”  <em>Aequorea</em>. <em>J. Cell. Comp. Physiol</em>.  v. 59, pp. 223–239.</li>
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      <li>(3) Shimomura, O., Chalfie, M. &amp; Tsien, R. Y. (2008) “<em>The discovery and development of the green fluorescent protein,  GFP.”  </em>The Nobel Prize in Chemistry.  Retrieved from the website <a href="http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/">http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/</a> </li>
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      <li>(4) Prasher, D., C.,  Eckenrode, V.,  K., Ward,W.,W., Prendergast, F., G. &amp; Cormier, M., J. (1992) “Primary  structure of the <em>Aequorea victoria </em>green-fluorescent protein” <em>Gene.,  v. </em>111, pp. 229-233. </li>
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      <li>(5) Tsien, R., Y. (1998)  “The Green Fluorescent Protein” <strong> </strong><em>Annual Review of  Biochemistry.  </em>v. 67, pp. 509–544 </li>
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    </ul>
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    <p class="STYLE6">&nbsp;</p>
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    <p class="STYLE6">Plasmid backbone:</p>
    <p class="STYLE6">Plasmid backbone:</p>
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                                    pSP-GM1  
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                    <p>pSP-GM1 <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#plasmid">(read more)</a></p>
    <p>&nbsp;</p>
    <p>&nbsp;</p>
    <p class="STYLE6">SNF1  (modified subunits):</p>
    <p class="STYLE6">SNF1  (modified subunits):</p>
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                                  Snf1 (alfa), Snf4 (gamma)
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                    <p>Snf1 (alfa), Snf4 (gamma) <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#SNF1">(read more)</a></p>
    <p>&nbsp;</p>
    <p>&nbsp;</p>
    <p class="STYLE6">FP  positions:</p>
    <p class="STYLE6">FP  positions:</p>
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                                  N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma
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                    <p>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>
    <p>&nbsp;</p>
    <p>&nbsp;</p>
    <p class="STYLE6">Primer  design:</p>
    <p class="STYLE6">Primer  design:</p>
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                                  Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C
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                    <p>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>
    <p>&nbsp;</p></td>
    <p>&nbsp;</p></td>
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    <p><span class="STYLE8">2010-07-07 </span><br>
    <p><span class="STYLE8">2010-07-07 </span><br>
      Adnan,  Katarina and Julia<br>
      Adnan,  Katarina and Julia<br>
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      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 where 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>
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      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 where 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>
        </p>
        </p>
    <p><span class="STYLE8">2010-07-08</span><br>
    <p><span class="STYLE8">2010-07-08</span><br>

Revision as of 11:49, 15 July 2010

Chalmers University of Technology

preliminary work & lab notes
 
 
preliminary
 
 

Fluorescent Proteins:

Green Fluorescent Proteins (read more)

 

Plasmid backbone:

pSP-GM1 (read more)

 

SNF1 (modified subunits):

Snf1 (alfa), Snf4 (gamma) (read more)

 

FP positions:

N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma (read more)

 

Primer design:

Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C (read more)

 

 
 
lab work by date
 
 

2010-07-05
Malin och Katarina
An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1.

2010-07-06
Malin, Adnan and Peidi
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.

2010-07-07
Adnan, Katarina and Julia
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 & 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 where very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.

2010-07-08
Malin, Julia and Peidi
The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.

2010-07-09
Malin and Julia
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.
Adnan and Katarina
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.

2010-07-12
Adnan and Kemal
We ran a miniprep to purify the plasmids containing ECFP and EYFP from E.coli. The purification was successful. We checked if we had the correct product by cutting the plasmids with EcoRI and PstI. The restriction enzyme cut plasmids were to be run on a gel the next day.

2010-07-13
Karl and Adnan
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.
Julia and Lokesh
We run 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.

 
pictures
 
 

gel_plasmid

PIC 1: Gel of plasmid (cut)
 
 
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