Team:Grenoble/Biology/Notebook/July/week 28

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iGEM Grenoble 2012

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Project

July

Week 27 • Week 28 • Week 29 • Week 30

Week 28: July 09th to 15th

Goal of the week:

We wanted to recover and amplify the biobricks involved in our genetic networks:
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • pLAC (100bp)
  • fha (80bp)
  • eCFP (800bp)
  • pLAC_RBS (120bp)
  • RsmA (200bp)
  • rsmY (170bp)
  • pSB1A3 (2400bp)
  • pSB4K5 (2400bp)
  • pSB3C5 (2400bp)

We also planned to realise the gibson assemblies for the first constructions.

Monday, July 09th:

Precultured cells are prepared:
  • Strains = BW25113 WT, BW25113 cya-, BW25113 cya-pAra/Bad and the strain transformed with pSB4K5
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight

Tuesday, July 10th:

For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.

We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
  • fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.
  • pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.
  • RBS_Cya from BW25113 WT precultured cells.
We did the PCR both with and without DMSO (Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO).

To separate (protocol) the PCR products, we prepared two gels:
  • a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)
  • a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)
Migration conditions = 50V during 1h15.
We used EtBr to reveal the DNA fragments.

photo_gel_4

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

  • Lane 1: DNA ladder 100bp (biolabs)
  • Lanes 2 and 3: fha1 PCR product
  • Lanes 4 and 5: RsmA PCR product
  • Lanes 6 and 7: rsmY PCR product
  • Lanes 8 and 9: fha1 PCR product (DMSO)
  • Lanes 10 and 11: RsmA PCR product (DMSO)
  • Lane 12: rsmY PCR product (DMSO)
  • Lane 13: DNA ladder 100bp (biolabs)

photo_gel_5
Migration result for the 0.8% TAE agarose gel (big fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lanes 2 and 3: RBS_Cya PCR product
Lanes 4 and 5: pSB1A3 PCR product
Lanes 6 and 7: pAra/Bad_RBS_GFP PCR product
Lanes 8 and 9: RBS_Cya PCR product (DMSO)
Lanes 10 and 11: pSBA13 PCR product (DMSO)
Lane 12: pAra/Bad_GFP PCR product (DMSO)
Lane 13: DNA ladder 1kb (biolabs)

There was a migration problem on the second gel (DNA ladder migration was not right) and we only saw primer dimer bands. There was a PCR condition problem.

Precultured cells were prepared:
  • Strains = BW25113 WT.
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

Wednesday, July 11th:

We did a miniprep (protocol) on three iGEM Grenoble 2011 strains:
  • fha1
  • RsmA
  • rsmY

We did a 15min digestion (protocol) by some restriction enzymes (XbaI and PstI) in order to check if there were the right plasmids in the strains.

To separate (protocol) the digestion products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 50V during 1h15.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_6
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100pb (biolabs)
Lanes 2 and 3: fha1 digestion product (XbaI)
Lanes 4 and 5: RsmA digestion product (XbaI)
Lanes 6 and 7: rsmY digestion product (XbaI)
Lanes 8 and 9: fha digestion product (pSTI)
Lanes 10 and 11: RsmA digestion product (pSTI)
Lanes 12 and 13: rsmY digestion product (pSTI)
Lanes 14 and 15: fha1 digestion product (XbaI-pSTI)
Lanes 16 and 17: RsmA digestion product (XbaI-pSTI)
Lanes 18 and 19: rsmY digestion product (XbaI-pSTI)
Lane 20: DNA ladder 100pb (biolabs)

We seen the DNA bands were at the wrong position, like there was no digestion. We thought the digestion problem occured during the heating step (we had a problem using the thermoblock).

Using iGEM 2012 biobricks we transformed (protocol) BW25113 WT cells. We obtained five transformed strains with five different biobricks:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • pSB3C5 plasmid
  • psB4K5 plasmid
  • psB1A3 plasmid

Precultured cells were prepared:
  • Strains = BW25113 cya- pAra/Bad.
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

Thursday, July 12th:

The transformation using pSB4K5 is the only one out of five transformations that showed results.

We did some PCRs with a HF Phusion enzyme (protocol) from miniprep (12/07/11) to amplify rsmY and fha1. We did the PCR both with and without DMSO.

To separate (protocol) the PCR products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 50V during 1h.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_7
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: fha1 PCR product
Lane 3: rsmY PCR product
Lane 4: fha1 PCR product (DMSO)
Lane 5: rsmY PCR product (DMSO)
Lane 6: DNA ladder 100bp (biolabs)
Lane 7 : empty

We saw no DNA bands at the right position, we thought there was still a PCR condition problem.

We did a miniprep (protocol) 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 some colony PCR with a HF Phusion enzyme (protocol) from iGEM Grenoble 2011 glycerol stock, to amplify: pLAC (fha1), pLAC (rsmY), pLAC_RBS, eCFP and RsmA.

To separate (protocol) the PCR products and the miniprep 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_8
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 2: DNA ladder 1kb (biolabs)
Lane 3: pSB4K5 miniprep product
Lane 4: plasmid with pAra/Bad miniprep product
Lane 5: empty
Lane 7: eCFP PCR product
Lane 8: pLAC (fha1) PCR product
Lane 9: pLAC_RBS PCR product
Lane 10: pLAC (rsmY) PCR product
Lane 11: RsmA PCR product
Lane 12: DNA ladder 100bp (biolabs)
Lane 13 : empty

The DNA bands corresponding to pLAC(fha1), pLAC_RBS pLAC(rsmY) and RsmA were at the expected positions. We thus decided to do the migration again in order to purify these PCR products.

We decided to fix the agarose percentage in our gels at 1.8% to separe the small fragments (length < 1000bp) and at 1.3% for biggest fragments (length > 1000bp).

Precultured cells were prepared:
  • Strains = BW25113 WT, pSB1A3 (iGEM Grenoble 2011) and pSB3C5 (iGEM Grenoble 2011).
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

Friday, July 13th:

To separate (protocol) the PCR products (PCRs 07/12), 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_9
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: eCFP PCR product
Lane 3: pLAC (fha1) PCR product
Lane 4: pLAC_RBS PCR product
Lane 5: pLAC (rsmY) PCR product
Lane 6: RsmA PCR product
Lane 7: empty
Lane 8: rsmY PCR product
Lane 9: fha1 PCR product
Lane 10: empty
Lane 11: rsmY PCR product
Lane 12: fha PCR product
Lane 13: DNA ladder 100bp (biolabs)

We realised a DNA extraction (protocol) from all the fragments except the fha1 PCR product (we didn’t see anything) and the eCFP PCR product (because its size did not correspond to the expected one: 800bp):
  • pLAC (fha1) 120713PP_PCR_009
  • pLAC_RBS 120713PP_PCR_010
  • pLAC (rsmY) 120713PP_PCR_011
  • RsmA 120713PP_PCR_012
  • rsmY 120713PP_PCR_013

We did a miniprep (protocol) on 2 strains (12/07/12) to recover pSB1A3 and pSB3C5.

We did some PCR with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB1A3, pSB3C5 and on another miniprep (12/07/12) to amplify pSB4K5. We did the PCR both with and without DMSO.

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_10
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB1A3 PCR product
Lane 3: pSB3C5 (cya) PCR product
Lane 4: pSB3C5 (RsmA) PCR product
Lane 5: pSB4K5 PCR product
Lane 6: pSB1A3 PCR product (DMSO)
Lane 7: pB3C5 (cya) PCR product (DMSO)
Lane 8: pSB3C5 (RsmA) PCR product (DMSO)
Lane 9: pSB4K5 PCR product (DMSO)
Lane 10: DNA ladder 1kb (biolabs)

We realised a DNA extraction (protocol) for all the fragments except the pSB4K5 PCR product (we didn’t see anything):
  • pSB1A3 120713PP_PCR_014
  • pSB3C5 (cya) 120713PP_PCR_015
  • pSB3C5 (RsmA) 120713PP_PCR_016

We realised two Gibson Assembly (protocol) to build two plasmids:
  • pSB1A3 120713PP_PCR_014 with pLAC (rsmY) 120713PP_PCR_011 and rsmY 120713PP_PCR_013
  • pSB3C5 (RsmA) 120713PP_PCR_016 with pLAC_RBS 120713PP_PCR_010 and RsmA 120713PP_PCR_012

Conclusion of the week:

We have achieved to amplify : pLAC (fha1) // pLAC (rsmY) // pLAC_RBS // RsmA // rsmY // pSB1A3 // pSB3C5 (Cya) // pSB3C5 (RsmA)

We tried our first Gibson Assemblies.

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