Team:Grenoble/Biology/Notebook/July/week 30

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July

Week 27 • Week 28 • Week 29 • Week 30

Week 30: July 23rd to 29th

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)
  • fha (80bp)
  • eCFP (800bp)
  • pSB4K5 (2400bp)

We also wanted to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.

Monday, July 23rd:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.

results_AND_Gate
strain used = BW25113 cya- transformed with pAra/Bad_RBS_GFP. The medium was complemented: 0.03% glucose & 0.03% acetate.

The experiments worked well, the "AND" gate worked as expected.

We did PCRs with HF Phusion enzyme (protocol) on miniprep in order to amplify pAra/Bad_RBS_GFP, and on colony to amplify RBS_Cya. We did the PCR both with and without DMSO.

To separate (protocol) the PCR products (12/07/23 and 12/07/20), 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_16

Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 80pb-10kb (fermentas)
  • Lane 2: pSB4K5 PCR product (DMSO)
  • Lane 3: fha1 PCR product (DMSO)
  • Lane 4: fha1 PCR product
  • Lane 5: pSB4K5 PCR product
  • Lane 6: pAra/Bad_RBS_GFP PCR product (DMSO)
  • Lane 7: pAra/Bad_RBS_GFP PCR product (DMSO)
  • Lane 8: RBS_Cya PCR product (DMSO)
  • Lane 9: RBS_Cya PCR product
  • Lane 10: pAra/Bad_RBS_GFP PCR product
  • Lane 11:pAra/Bad_RBS_GFP PCR product
  • Lane 12: DNA ladder 80bp-10kb (fermentas)

We saw primer dimer bands and the bands corresponding to the amplified fha1 sequence (lanes 3 & 4). For the other, there was a PCR condition problem.

We realised a DNA extraction (protocol) from the two fha PCR products 120723AM_PCR_020.

We did touchdown PCRs with HF Phusion enzyme (protocol) on digestion product (eCFP E0022 and E0422) (12/07/20) in order to amplify eCFP. We did the PCR both with and without DMSO.

Using iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4K5.

Tuesday, July 24th:

To separate (protocol) the PCR products (12/07/23), 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_17

Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 80bp-10kb (fermentas)
  • Lane 2: E0022 PCR product
  • Lane 3: E0022 PCR product (DMSO)
  • Lane 4: E0422 PCR product
  • Lane 5: E0422 PCR product (DMSO)
  • Lane 6: DNA ladder 80bp-10kb (fermentas)

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

The transformation with pSB4K5 (12/07/23) showed result (growth on plates).

We did some digestions (protocol) on miniprep (12/07/19) to recover eCFP (E0022). The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate (protocol) 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_18

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: pSB4K5 digestion product
  • Lane 4: DNA ladder 80bp-1kb (fermentas)

The digestion worked, we saw DNA bands at the expected positions.

We did PCR with HF Phusion enzyme (protocol) from one of these digestion product (eCFP E0022) in order to amplify eCFP and a colony PCR to amplify pSB4K5.

To separate (protocol) 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.

There was no result (data not shown).

We didn’t achieve to amplify pSB4K5. We decided to change it, we chose pSB4C5 instead. So we had to change pSB3C5, we chose pSB3T5.

With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4C5.

We did an overlapping PCR with HF Phusion enzyme (protocol) on the digestion product (eCFP E0022) in order to amplify eCFP and on miniprep to amplify fha1.

Wednesday, July 25th:

To separate (protocol) the PCR products (12/07/24), we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

There was a migration problem (data not shown).

The transformation with pSB4C5 (12/07/24) was successful (growth on plates). We decided to relaunch it in fresh liquid LB.

We did some glycerol stocks:
  • GA: pLAC_RBS_RsmA 120725GH_001
  • pSB4K5 120725GH_002
  • E0422 (eCFP) 120725GH_003
  • LuxI 120725GH_004
  • E0022 (eCFP) 120725GH_005
  • LuxR 120725GH_006
  • pLux 120725GH_007
  • I13601 120725GH_008

We did a colony PCR with GoTaq enzyme (protocol) in order to amplify pSB4C5.

To separate (protocol) the PCR products (07/24), 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_19

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 PCR product
  • Lane 3: DNA ladder 1kb (biolabs)

We concluded that the pSB4C5 amplification worked well, so we decided to do the PCR again in order to do a DNA extraction.

Thursday, July 26th:

We did a glycerol stock of the strain transformed with pSB4C5 120726AM_009.

We did a colony PCR withs HF Phusion Enzyme (protocol) in order to amplify pSB4C5.

We did a miniprep (protocol) on the transformed strain with pSB4K5, on which we wanted to recover pSB4K5.

We did some digestions (protocol) on this miniprep, in order to check if pSB4K5 was ok. The digestion was realised with two restriction enzymes : XbaI and PstI during 10 minutes.

To separate (protocol) the PCR products (12/07/24), 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_20

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: pSB4K5 without digestion
  • Lanes 4 and 5: pSB4K5 digestion product
  • Lane 6: empty lane
  • Lanes 7 and 8: pSB4C5 PCR product
  • Lane 9: DNA ladder 1kb (biolabs)

We concluded that the digestion worked well (we seen the expected DNA bands), and so was the PCR on pSB4C5.

We realised a DNA extraction (protocol) on the two pSB4C5 PCR products 120726AM_PCR_021.

We didn’t achieve to amplify eCFP, we decided to change it. We chose mcherry and GFP.

We transformed (new protocol) BW25113 WT competent cells (protocol) with biobricks from iGEM 2012 kit :
  • BBa_I763011 (GFP)
  • BBa_J06702 (mcherry) (in duplicate : in LB with Ampicillin and in LB with Ampicillin and Kanamycin)
  • pSB1A3
  • BBa_R0082 (pompC)
  • BBa_J23119 (pConst)

Friday, July 27th:

The five transformations (12/07/26) were successful (growth on the expected plates). We did glycerol stocks of:
  • BBa_J06702 (mcherry in LB with Ampicillin and Kanamycin) 120727GH_010
  • pSB1A3 120727GH_011
  • BBa_J23119 (pConst) 120727GH_012
  • BBa_R0082 (pOmpC) 120727GH_013
  • BBa_J06702 (mcherry in LB with Ampicillin) 120727GH_014
  • BBa_I763011 (GFP) 120727GH_015

We did some colony PCRs with HF Phusion enzyme (protocol) in order to amplify RBS_Cya (on BW25113 WT strain), pAra/Bad_RBS_GFP (on BW25113 Cya- pAra/Bad) and pSB4C5 (on 12/07/25 colony).

To separate (protocol) 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_21

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: pAra/Bad_RBS_GFP PCR product
  • Lanes 4 and 5: RBS_Cya PCR product
  • Lanes 6 and 7: pSB4C5 PCR product
  • Lane 8: DNA ladder 1kb (biolabs)

The pSB4C5 amplification didn’t work. However, the RBS_Cya and pAra/Bad_RBS_GFP amplications worked.

We realised a DNA extraction (protocol) from the two pAra/Bad_RBS_GFP PCR products 120727AM_PCR_023 and the two Cya PCR products 120727AM_PCR_022.

Conclusion of the week:

We replaced pSB4K5 with pSB4C5 and pSB3C5 with pSB3T5. We also replaced eCFP with mcherry or 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.

The "AND" gate worked as expected.

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