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

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iGEM Grenoble 2012

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Project

July

week 27 week 28 week 29 week 30

Week 29: July 16th to 22nd

Goal of the week:

We wanted to 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)
Lanes 2 and 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 18th:

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 19th:

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)

We concluded that the Gibson Assembly (pLAC_RBS_RsmA on pSB1A3) worked. The digestion result is the expected one.

We did PCRs with HF Phusion enzyme (protocol) from minipreps in order to amplify pAra/Bad_RBS_GFP (12/07/17), eCFP (E0022 et E0422) (12/07/19). And a colony PCR to amplify RBS_Cya. We did the PCR both with and without DMSO.

To separate the PCR products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_14
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: eCFP (E0022) PCR product
Lane 3: eCFP (E0022) PCR product (DMSO)
Lane 4: RBS_Cya PCR product
Lane 5: RBS_Cya PCR product (DMSO)
Lane 6: pAra/Bad_GFP PCR product 1
Lane 7: pAra/Bad_GFP PCR product 1 (DMSO)
Lane 8: pAra/Bad_GFP PCR product 2
Lane 9: pAra/Bad_GFP PCR product 2 (DMSO)
Lane 10: eCFP (E0422) PCR product
Lane 11: eCFP (E0422) PCR product (DMSO)
Lane 12: DNA ladder 1kb (biolabs)

We saw primer dimer bands and the bands corresponding to the amplified plasmids which brought eCFP (E0022 & E0422). There was a PCR condition problem.

One transformation (12/07/18) showed result during the day: rsmY. We decided to relaunch it in fresh LB medium + antibiotics.

Friday, July 20th:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/18) with Gibson Assembly product (RsmA and rsmY).

In order to check the products of Gibson Assembly (RsmA and rsmY) we launched digestion experiments on these miniprep products, using restriction enzymes (EcoRI, BamHI, XbaI and SpeI) during 10 minutes. We also launched digestion experiments on the eCFP miniprep products (12/07/19), using the same restriction enzymes, in order to recover the eCFP coding sequence.

To separate these 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_15
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: E0422 digestion product
Lane 3: E0022 digestion product
Lane 4: GA (RsmA) miniprep product
Lane 5: GA (rsmY) miniprep product
Lane 6: DNA ladder 1kb (biolabs)
Lane 7: DNA ladder 100bp (biolabs)
Lane 8: GA (RsmA) digestion product
Lane 9: GA (rsmY) digestion product
Lane 10: DNA ladder 100bp (biolabs)

We realised a DNA extraction (protocol kit: Nucleospin extract II) on the bands corresponding to the eCFP coding sequence (E0422 and E0022) from digestion products. (digestion product codes = 120720AM_DIG_018 & 120720AM_DIG_19).
On these extractions, we did PCRs with HF Phusion enzyme (protocol) in order to amplify eCFP. We did the PCR both with and without DMSO.

To separate the PCR products and the digestion products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_15
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: E0022 digestion product
Lane 3: E0422 digestion product
Lane 4: E0022 PCR product
Lane 5: E0022 PCR product (DMSO)
Lane 6: E0422 PCR product
Lane 7: E0422 PCR product (DMSO)
Lane 8: DNA ladder 1kb (biolabs)

We saw primer dimer bands. There was a PCR condition problem.

We did PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4K5 and fha from miniprep. We did the same PCR twice: one with DMSO and one without.

Conclusion of the week:

We have achieved our first Gibson Assembly : pLAC_RBS_RsmA on pSB3C5.

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