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

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iGEM Grenoble 2012

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July

week 27 week 28 week 29 week 30

Week 30: July 23rd to 29th

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

Monday, July 23rd:

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 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 (lane 2 & 5). For the other, there was a PCR condition problem.

We realised a DNA extraction (protocol kit: Nucleospin extract II) from the two fha PCR products (PCR product code = 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 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 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 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 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 :
  • RsmA (GA) (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 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_18
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)

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