Team:Grenoble/Biology/Notebook/July/week 29

From 2012.igem.org

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In order to reveal the DNA fragments, we used EtBr.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
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<center><img src="https://static.igem.org/mediawiki/2012/5/51/120719.jpg" alt="photo_gel_13"/></center>
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<center><table>
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<tr><center><img src="https://static.igem.org/mediawiki/2012/5/51/120719.jpg" alt="photo_gel_13"/></center>
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<tr><u>Migration result for a 1.8% 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 100bp (biolabs)<br/>
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Lane 2: GA (RsmA) digestion product (XbaI)<br/>
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Lane 3: GA (RsmA) digestion product (SpeI)<br/>
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Lane 4: GA (RsmA) digestion product (XbaI & SpeI)<br/>
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Lane 5: DNA ladder 100bp (biolabs)<br/></tr>
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</table></center>

Revision as of 06:36, 7 August 2012

iGEM Grenoble 2012

Project
JULY week 27 week 28 week 29 week 30

Week 29: July 16th to July 22nd

Goal of the week

test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks :
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • fha1 (80bp)
  • eCFP (800bp)
  • pSB4K5 (2400bp)

Monday, July 16th:

Precultured cells are prepared:
  • Strains = BW25113 WT and BW25113 cya- pAra/Bad
  • Conditions = Conditions = LB liquid medium, 37°C, 200rpm, overnight

Tuesday, July 17th:

Using iGEM 2012 biobricks and the Gibson Assembly product we transformed (protocol) BW25113 WT cells. We obtained four transformed strains with:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • 120713PP_GA_001 (rsmY)
  • 120713PP_GA_002 (RsmA)

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains, on which we wanted to recover pSB4K5 and the plasmid with pAra/Bad_RBS_GFP:
  • transformed strain with pSB4K5
  • BW25113 cya- pAra/Bad

We did PCRs with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB4K5, and from colony to amplify pLAC (fha), pLAC (rsmY), pLAC_RBS, fha and pSB4K5. We did the PCR both with and without DMSO.

To separate the PCR products and the miniprep products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_11
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2-3: the deposits have failed
Lane 4: pSB4K5 colony PCR product (DMSO)
Lane 5: pSB4K5 colony PCR product
Lane 6: pSB4K5 PCR (miniprep) product (DMSO)
Lane 7: pSB4K5 PCR (miniprep) product
Lane 8: DNA ladder 1kb (biolabs)
Lane 9: DNA ladder 100bp (biolabs)
Lane 10: pLAC (fha1) PCR product (DMSO)
Lane 11: pLAC (fha1) PCR product
Lane 12: pLAC_RBS PCR product (DMSO)
Lane 13: pLAC_RBS PCR product
Lane 14: pLAC (rsmY) PCR product (DMSO)
Lane 15: pLAC (rsmY) PCR product
Lane 16: fha1 PCR product (DMSO)
Lane 17: fha1 PCR product
Lane 18: empty
Lane 19: pSB4K5 miniprep product
Lane 20: plasmid with pAra/Bad_RBS_GFP miniprep product

We achieved to amplify pLAC (fha1), pLAC (rsmY) and pLAC_RBS. We didn’t achieve to amplify pSB4K5 and fha1.

Wednesday, July 17th:

We did PCRs with HF Phusion enzyme (protocol) on a miniprep (12/07/17) in order to amplify pAra/Bad_RBS_GFP, on colony (iGEM Grenoble 2011) to amplify fha1, and on glycerol stock (BW25113 WT) to amplify RBS_Cya. We did the PCR both with and without DMSO.

To separate the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_12
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: pAra/Bad_RBS_GFP PCR product (1μL/DMSO)
Lane 3: pAra/Bad_RBS_GFP PCR product (2μL/DMSO)
Lane 4: pAra/Bad_RBS_GFP PCR product (1μL)
Lane 5: pAra/Bad_RBS_GFP PCR product (2μL)
Lane 6: RBS_Cya PCR product (DMSO)
Lane 7: RBS_Cya PCR product
Lane 8: fha1 PCR product (DMSO)
Lane 9: fha1 PCR product
Lane 10: DNA ladder 100bp (biolabs)

We didn’t see anything. The PCR didn’t work.

We did PCRs with HF Phusion enzyme (protocol) on miniprep (12/07/17) in order to amplify pSB4K5, and on 2 colonies (iGEM Grenoble 2011) to amplify fha1. We did the PCR both with and without DMSO.

To separate the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

No result. (data not shown)

Using biobricks from the 2012 iGEM kit and our Gibson Assemblies product we transformed (new protocol) BW25113 WT competent cells (protocol). We obtained eight transformed strains with:
  • lux pR (BBa_R0062)
  • LuxI (BBa_C0061)
  • LuxR (BBa_I0462)
  • eCFP (BBa_E0022)
  • eCFP_ (BBa_E0422)
  • pLAC_RBS (BBa_I13601)
  • 120713PP_GA_001 (rsmY)
  • 120713PP_GA_002 (RsmA)

Thursday, July 18th:

Six transformations (12/07/18) out of eight showed results: RsmA, lux pR, LuxI, LuxR, E0422, E0022.

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on these transformed strains (12/07/18): RsmA, lux pR, LuxI, LuxR, E0422, E0022.

We also did some digestions (protocol) in order to check if the Gibson Assembly product, 120713PP_GA_002 (RsmA) is the right one. The digestions were realised with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate the digestion products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_13
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: GA (RsmA) digestion product (XbaI)
Lane 3: GA (RsmA) digestion product (SpeI)
Lane 4: GA (RsmA) digestion product (XbaI & SpeI)
Lane 5: DNA ladder 100bp (biolabs)