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| - | {{:Team:Grenoble/Templates/Biology}}
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| - | <html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">
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| - | <body id="Biology">
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| - | <section>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July">week 27</a>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_28">week 28</a>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_29">week 29</a>
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| - | <a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/June/week_30">week 30</a>
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| - | <br/>
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| - | <br/>
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| - | <h1> Week 28: July 09<span class="exposant">th</span> to July 15<span class="exposant">th</span> </h1>
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| - | <h2> Goal of the week </h2>
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| - | we wanted to recover and amplify the biobricks involved in our genetic networks:
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| - | <ul>
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| - | <li>pAra/Bad_RBS_GFP (1300bp)</li>
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| - | <li>RBS_Cya (2600bp)</li>
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| - | <li>pLAC (100bp)</li>
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| - | <li>fha (80bp)</li>
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| - | <li>eCFP (800bp)</li>
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| - | <li>pLAC_RBS (120bp)</li>
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| - | <li>RsmA (200bp)</li>
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| - | <li>rsmY (170bp)</li>
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| - | <li>pSB1A3 (2400bp)</li>
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| - | <li>pSB4K5 (2400bp)</li>
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| - | <li>pSB3C5 (2400bp)</li>
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| - | </ul>
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| - | We also planned to realise the gibson assemblies for the first constructions.
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| - | <br/><br/>
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| - | <h2> Monday, July 09<span class="exposant">th</span>:</h2>
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| - | Precultured cells are prepared:
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| - | <ul>
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| - | <li>Strains = BW25113 WT and BW25113 cya- pAra/Bad </li>
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| - | <li>Conditions = LB liquid medium, 37°C, 200rpm, overnight </li>
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| - | </ul>
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| - | <br/>
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| - | <h2>Tuesday, July 10<span class="exposant">th</span>:</h2>
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| - | For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.<br/>
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| - | We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
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| - | <ul>
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| - | <li>fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.</li>
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| - | <li>pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.</li>
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| - | <li>RBS_Cya from BW25113 WT precultured cells.</li>
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| - | </ul>
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| - | We did the PCR both with and without DMSO (Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO).<br/>
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| - | <br/>
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| - | To separate the PCR products, we prepared two gels:
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| - | <ul>
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| - | <li>a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)</li>
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| - | <li>a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)</li>
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| - | </ul>
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| - | <br/>Migration conditions = 50V during 1h15.<br/>
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| - | We used EtBr to reveal the DNA fragments.<br/>
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| - | <center><img src="https://static.igem.org/mediawiki/2012/b/b6/120710.jpg" alt="photo_gel_4"/></center>
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| - | <u>Migration result for the 1.3% TAE agarose gel (small fragments)</u><br/>
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| - | <i>(the DNA ladder scale is in kb)</i><br/>
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| - | Lane 1: DNA ladder 100bp (biolabs)<br/>
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| - | Lane 2 and 3: fha1 PCR product<br/>
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| - | Lane 4 and 5: RsmA PCR product<br/>
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| - | Lane 6 and 7: rsmY PCR product<br/>
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| - | Lane 8 and 9: fha1 PCR product (DMSO)<br/>
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| - | Lane 10 and 11: RsmA PCR product (DMSO)<br/>
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| - | Lane 12: rsmY PCR product (DMSO)<br/>
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| - | Lane 13: DNA ladder 100bp (biolabs)<br/>
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| - | <br/>
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| - | <center><img src="https://static.igem.org/mediawiki/2012/4/4e/120710_%282%29.jpg" alt="photo_gel_5"/></center>
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| - | <u>Migration result for the 0.8% TAE agarose gel (big fragments)</u><br/>
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| - | <i> (the DNA ladder scale is in kb)</i><br/>
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| - | Lane 1: DNA ladder 1kb (biolabs)<br/>
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| - | Lane 2 and 3: RBS_Cya PCR product<br/>
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| - | Lane 4 and 5: pSB1A3 PCR product<br/>
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| - | Lane 6 and 7: pAra/Bad_RBS_GFP PCR product<br/>
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| - | Lane 8 and 9: RBS_Cya PCR product (DMSO)<br/>
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| - | Lane 10 and 11: pSBA13 PCR product (DMSO)<br/>
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| - | Lane 12: pAra/Bad_GFP PCR product (DMSO)<br/>
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| - | Lane 13: DNA ladder 1kb (biolabs)<br/>
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| - | <br/>
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| - | There was a migration problem on the second gel (DNA ladder migration was not right) and we only saw primer dimer bands. There was a PCR condition problem.<br/>
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| - | <br/>
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| - | Precultured cells were prepared:
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| - | <ul>
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| - | <li>Strains = BW25113 WT.</li>
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| - | <li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</li>
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| - | </ul>
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| - | <br/>
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| - | <h2> Wednesday, July 11<span class="exposant">th</span>:</h2>
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| - | We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on three iGEM Grenoble 2011 strains:
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| - | <ul>
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| - | <li>fha1</li>
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| - | <li>RsmA</li>
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| - | <li>rsmY</li>
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| - | </ul>
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| - | <br/>
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| - | We did a 15min digestion (protocol) by some restriction enzymes (XbaI and PstI) in order to check if there were the right plasmids in the strains.<br/>
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| - | <br/>
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| - | To separate the digestion products, we prepared a 1.3% TAE agarose gel.<br/>
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| - | Migration conditions = 50V during 1h15.<br/>
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| - | In order to reveal the DNA fragments, we used EtBr.<br/>
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| - | <center><img src="https://static.igem.org/mediawiki/2012/3/3f/120711.jpg" alt="photo_gel_6"/></center>
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| - | <u>Migration result for a 1.3% TAE agarose gel</u><br/>
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| - | <i>(the DNA ladder scale is in kb)</i><br/>
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| - | Lane 1: DNA ladder 100pb (biolabs)<br/>
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| - | Lane 2 and 3: fha1 digestion product (XbaI)<br/>
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| - | Lane 4 and 5: RsmA digestion product (XbaI)<br/>
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| - | Lane 6 and 7: rsmY digestion product (XbaI)<br/>
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| - | Lane 8 and 9: fha digestion product (pSTI)<br/>
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| - | Lane 10 and 11: RsmA digestion product (pSTI)<br/>
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| - | Lane 12 and 13: rsmY digestion product (pSTI)<br/>
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| - | Lane 14 and 15: fha1 digestion product (XbaI-pSTI)<br/>
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| - | Lane 16 and 17: RsmA digestion product (XbaI-pSTI)<br/>
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| - | Lane 18 and 19: rsmY digestion product (XbaI-pSTI)<br/>
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| - | Lane 20: DNA ladder 100pb (biolabs)<br/>
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| - | <br/>
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| - | We seen the DNA bands were at the wrong position, like there was no digestion. We thought the digestion problem occured during the heating step (we had a problem using the thermoblock).<br/>
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| - | <br/>
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| - | Using iGEM 2012 biobricks we transformed (protocol) BW25113 WT cells. We obtained five transformed strains with five different biobricks:
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| - | <ul>
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| - | <li>BBa_I13601: pLAC_RBS</li>
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| - | <li>BBa_E0422: eCFP </li>
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| - | <li>pSB3C5 plasmid </li>
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| - | <li>psB4K5 plasmid </li>
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| - | <li>psB1A3 plasmid</li>
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| - | </ul>
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| - | Precultured cells were prepared:
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| - | <ul>
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| - | <li>Strains = BW25113 cya- pAra/Bad.</li>
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| - | <li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</li>
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| - | </ul>
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| - | </section>
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| - | </body>
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| - | </html>
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| - | {{:Team:Grenoble/script}}
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| - | {{:Team:Grenoble/menu}}
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