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

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iGEM Grenoble 2012

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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

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