Team:Grenoble/Biology/Notebook/August

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iGEM Grenoble 2012

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Project

August

Week 31 • Week 32 • Week 33 • Week 34 • Week 35

Week 31: July 30th to August 05th

Goal of the week:

We wanted to recover and amplify some biobricks involved in our genetic networks:
  • pSB4C5 (2400bp)
  • pompC (100bp)
  • mcherry (900bp)
  • eCFP (800bp)
  • GFP (800bp)

Tuesday, July 31th:

We did a miniprep (protocol) on the transformed strain with pSB4C5 (12/07/25), on which we wanted to recover pSB4C5.

We did some colony PCRs (on 12/07/25 colony) and PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 60°C.

To separate (protocol) 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_22

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 1kb (biolabs)
  • Lane 2: pSB4C5 (miniprep) PCR product (DMSO)
  • Lane 3: pSB4C5 (miniprep) PCR product (DMSO)
  • Lane 4: pSB4C5 (miniprep) PCR product
  • Lane 5: pSB4C5 (miniprep) PCR product
  • Lane 6: pSB4C5 (colony) PCR product (DMSO)
  • Lane 7: pSB4C5 (colony) PCR product (DMSO)
  • Lane 8: pSB4C5 (colony) PCR product
  • Lane 9: pSB4C5 (colony) PCR product
  • Lane 10: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 65°C.

We did some digestions (protocol) on miniprep (pSB4C5) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate (protocol) the PCR products and 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_23

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 1kb (biolabs)
  • Lane 2: pSB4C5 digestion product
  • Lane 3: pSB4C5 digestion product
  • Lane 4: pSB4C5 (miniprep) PCR product (DMSO)
  • Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
  • Lane 6: pSB4C5 (miniprep) PCR product
  • Lane 7: pSB4C5 (miniprep) PCR product
  • Lane 8: pSB4C5 (colony) PCR product (DMSO)
  • Lane 9: pSB4C5 (colony) PCR product (DMSO)
  • Lane 10: pSB4C5 (colony) PCR product
  • Lane 11: pSB4C5 (colony) PCR product
  • Lane 12: DNA ladder 1kb (biolabs)

For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.
The digestion worked well, we realised a DNA extraction (protocol) from the two digestion products 120731AM_DIG_027.

Wednesday, August 01st:

We did a touchdown PCR (from 70°C to 60°C) from miniprep (12/07/31) with HF Phusion enzyme (protocol) in order to amplify pSB4C5.

To separate (protocol) 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_24

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 1kb (biolabs)
  • Lane 2: pSBAC5 (digestion) PCR product
  • Lane 3: pSB4C5 (digestion) PCR product (DMSO)
  • Lane 4: pSB4C5 (miniprep) PCR product
  • Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
  • Lane 6: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We realised eight Gibson Assemblies (protocol) to build four plasmids:
  • pSB4C5 (fha1) 120726AM_PCR_021 with pLAC (fha1) 120713PP_PCR_009, fha1 120723AM_PCR_020 and eCFP 120720_DIG_019
  • pSB1A3 120713PP_PCR_014 with pLAC (rsmY) 120713PP_PCR_011 and rsmY 120713PP_PCR_013
  • pSB3C5 (Cya) 120713PP_PCR_015 with pAra/Bad_RBS_GFP 120727AM_PCR_023 and RBS_Cya 120727AM_PCR_022
  • pSB4C5 120731AM_DIG_027 with pAra/Bad_RBS_GFP 120727AM_PCR_023 and RBS_Cya 120727AM_PCR_022

For each plasmid construct, we did two Gibson Assemblies (protocol).
We transformed ((new protocol) BW25113 WT competent cells (protocol) with the GA products:
pLAC_fha1_eCFP and pLAC_rsmY.
We transformed ((new protocol) BW25113 Cya- competent cells (protocol) with the GA products:
pAra/Bad_RBS_GFP_RBS_Cya.

We did some minipreps (protocol) on the transformed strains (12/07/25) with pOmpC, mcherry, eCFP and pSB4C5 ; in order to set up the test on the receptor with a reporter.

Then, we did some digestions (protocol) on these minipreps. The digestions were achieved with two restriction enzymes:
  • pOmpC with EcoRI and SpeI during 10 minutes
  • mcherry and eCFP with XbaI and SpeI during 10 minutes
  • pSB4C5 with EcoRI and PstI during 10 minutes

To separate (protocol) 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_25

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 80pb-10kb (fermentas)
  • Lane 2: pOmpC digestion product
  • Lane 3: pOmpC digestion product
  • Lane 4: DNA ladder 80pb-10kb (fermentas)
  • Lane 5: eCFP digestion product
  • Lane 6: eCFP digestion product
  • Lane 7: mcherry digestion product
  • Lane 8: mcherry digestion product
  • Lane 9: pSB4C5 digestion product
  • Lane 10: pSB4C5 digestion product
  • Lane 11: DNA ladder 1kb (biolabs)

The digestion worked well, we realised a DNA extraction (protocol) from the eCFP, mcherry and pSB4C5 digestion products.

Thursday, August 02nd:

We did a miniprep (protocol) on the transformed strain (12/07/25) with pOmpC.

Then, we did a digestion (protocol) on this miniprep. The digestions were achieved with two restriction enzymes: EcoRI and SpeI during 10 minutes.

To separate (protocol) 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_26

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 80pb-10kb (fermentas)
  • Lane 2: pOmpC miniprep product
  • Lane 3: pOmpC miniprep product
  • Lane 4: pOmpC digestion product
  • Lane 5: pOmpC digestion product
  • Lane 6: DNA ladder 80pb-10kb (fermentas)

There was a problem with the miniprep or the transformation, we didn't see any DNA bands.

With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pOmpC.

We decided to relaunch the transformed strains with the GA products (12/08/01) in fresh liquid LB.

Friday, August 03rd:

We did some minipreps (protocol) on the transformed strains with the GA products.

We did some glycerol stocks:
  • GA: pLAC_rsmY (pSB1A3) 1 120803GH_016
  • GA: pLAC_fha1_eCFP (pSB4C5) 1 120803GH_017
  • GA: pLAC_fha1_eCFP (pSB4C5) 2 120803GH_018
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 120803GH_019
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 120803GH_020
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 120803GH_021
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 120803GH_022
  • GA: pLAC_rsmY (pSB1A3) 2 120803GH_023
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 120803GH_024
  • GA: pLAC_rsmY (pSB1A3) 3 120803GH_025
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 120803GH_026
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 5 120803GH_027

We decided to replace eCFP with mcherry, then we had to design the construction pLAC_fha1_mcherry and to design the primers used for this construction.
We also designed the primers used for the receptor construction: pCons(BBa_J23119)_RBS_Tap/EnvZ.

Conclusion of the week:

We replaced eCFP with mcherry GFP.

We have achieved to amplify pAra/Bad_RBS_GFP // RBS_Cya // fha // pSB4C5 (fha) and we began the experiments in order to recover pOmpC (for the detection module) and pConst.

We decided to focus on the system with the pAra/Bad "AND" gate.

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