Team:Grenoble/Biology/Notebook/July

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iGEM Grenoble 2012

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Project

July

Week 27 • Week 28 • Week 29 • Week 30

Week 27: July 02nd to 08th

Goal of the week:

We wanted to test the PCR conditions, 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)

Tuesday, July 03rd:

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

Wednesday, July 04th:

We did PCRs on colony with a GoTaq 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.

Using iGEM 2012 biobricks we transformed BW25113 WT cells (protocol). We obtained four transformed strains with four different biobricks:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • pSB3C5 plasmid
  • pSB4K5 plasmid

Thursday, July 5th:

The transformations (12/07/04) showed no growth on LB Agar plate + antibiotics.

In order to separate (protocol) the PCR products (12/07/04), we prepared two gels:
  • a 1.3% Tris base Acetic acid EDTA (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 = 100V during 35 min.
In order to reveal the fragments, we used Ethidium Bromide (EtBr).

photo_gel_1

Migration results 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: not iGEM PCR products
  • Lanes 4 and 5: RsmA PCR product
  • Lanes 6 and 7: fha1 PCR product
  • Lanes 8 and 9: rsmY PCR product
  • Lane 10: DNA ladder 100bp (biolabs)

photo_gel_2

Migration results 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: not iGEM PCR products
  • Lanes 4 and 5: pSB1A3 PCR product
  • Lanes 6 and 7: RBS_Cya PCR product
  • Lanes 8 and 9: pAra/Bad_RBS_GFP PCR product
  • Lane 10: DNA ladder 1kb (biolabs)

There is no visible migration on the second gel (big fragments). We realized a DNA extraction (protocol) from the three small fragments:
  • fha1 120705PP_PCR_001 // 120705PP_PCR_002
  • RsmA 120705PP_PCR_003 // 120705PP_PCR_004
  • rsmY 120705PP_PCR_005 // 120705PP_PCR_006

Precultured cells are prepared:
  • Strains = iGEM Grenoble 2011 glycerol stock with fha, RsmA and rsmY.
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

We did colony PCR with a GoTaq enzyme (protocol) to amplify:
  • RBS_Cya from BW25113 WT strain
  • pSB1A3 from iGEM 2011 glycerol stock

To separate (protocol) the PCR products, we prepared a 0.8% TAE agarose gel.
Migration conditions = 50V during 1h35.
The DNA fragments were revealed with EtBr.

photo_gel_3

Migration results for a 0.8% TAE agarose gel
(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: not iGEM PCR product
  • Lanes 6 and 7: not iGEM PCR product
  • Lanes 8 and 9: pSB1A3 PCR product
  • Lane 10: DNA ladder 1kb (biolabs)

There is no visible migration on the gel, we only saw primer dimer bands. There was a PCR condition problem.

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

Friday, July 6th:

As our PCR results were not conclusive, we decided to test the PCR conditions.

We did a miniprep (protocol) on BW25113 cya- pAra/Bad strain, in order to recover the plasmid with pAra/Bad_RBS_GFP on it.

To test the PCR conditions, we separated the sample in two groups (to check the Green buffer effect) subdivised into five samples according to the amount of template DNA:
  • 1st group: PCR with GoTaq (protocol) and Green Buffer 5X
  • 2nd group: PCR with GoTaq (protocol) and Colorless Buffer 5X (+ loading dye for the migration)

For each group, there were five samples (different concentration of template DNA):
  • 1st with 1μL of DNA template (= plasmid from the miniprep)
  • 2nd with 2μL of DNA template
  • 3rd with 3μL of DNA template
  • 4th with 4μL of DNA template
  • 5th with 0μL of DNA template


On the same plasmid (from the miniprep to check its efficiency), we separated the sample in three groups ; each group is subdivised into five samples to test the different amount of template DNA:
  • the 1st: no digestion
  • the 2nd: one hour digestion (protocol) by a restriction enzyme (=XbaI)
  • the 3rd: 10 min digestion (protocol) by a restriction enzyme (=XbaI)

For each group, there were five samples with different concentration of DNA:
  • 1st with 1μL of DNA template (= plasmid from the miniprep)
  • 2nd with 2μL of DNA template
  • 3rd with 3μL of DNA template
  • 4th with 4μL of DNA template
  • 5th with 0μL of DNA template

To separate (protocol) the PCR products and the digestion products, we prepared a 0.8% TAE agarose gel.
Migration conditions = 50V during 1h35.
The DNA fragments were revealed with EtBr.

There was no visible migration on the gel (data not shown).

Conclusion of the week:

As we had no conclusive results, we have decided to change the protocols.

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