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Lab Work - 11/09/09

Metal Sensing Team

Introduction and Outline

So far we have transformed DH5-alpha E. coli cells with two synthesized BioBricks, which are the cotC-GFP-smtA and the kinA BioBricks. The cotC-GFP-smtA BioBrick is involved within the metal sensing sub-project whereas the kinA BioBrick is involved with the Stochastic Switch sub-project. The Metal Sensing team have since taken these transformant colonies and used them to set up LB+kanamycin cultures for mini-preps and midi-preps; these were then left overnight in the orbital shaking incubator and are awaiting processing today. However there are a range of tasks which need to be accomplished today; these are listed below:

• Prepare Mini-preps of the kinA and cotC-GFP-smtA transformants
  • Carry out the mini-prep processes
  • Analyse the mini-prep DNA by treating with restriction enzymes.
• PCR the cotC-GFP-smtA BioBrick with pMK15 and pMK16
  • Set up the PCR
  • Run the PCR for the correct duration
  • Analyse PCR products through DNA gel electrophoresis
  • Clean up PCR products if PCR reaction proves to be successful
• Midi-prep cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3
  • Carry out the five midi-preps.
  • Quantify the midi-prep samples
  • Run the midi-prep samples on agarose gel through DNA gel electrophoresis
• Run products from a PCR reaction involving arsR and cadA DNA
  • Run PCR products on gel
  • If successful, clean up fragments, run on gel again, cut with restriction enzymes NheI and BamHI, clean and then ligate together fragments.

Procedure

The following procedures were carried out:

Freeze the kinA and cotC-GFP-smtA transformant cells

Instead of preparing mini-prep samples from the two groups of transformant E.coli cells (i.e. kinA transformed E.coli cells and cotC-GFP-smtA transformed cells), we instead took the decision to use the inoculated 5ml LB tubes in freezing the cells. Analysis of the midi-prep samples will be used to determine whether the BioBricks have been truly transformed - this decision will both save time and also provide a back-up stock of cells for future work. This procedure was carried out by James using the protocol

PCR the cotC-GFP-smtA BioBrick with pMK15 and pMK16

Midi-prep cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3

Run products from a PCR reaction involving arsR and cadA DNA

Results

We took the decision to run 4 sets of DNA samples on 0.8% agarose gel - this includes the midi-prep samples (i.e. cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3), products from a PCR reaction involving cotC, futher midi-preps of pMUTIN4 and digests from midi-prepped DNA the Stochastic Switch team have prepared (i.e. sac, ara and sleB) The resulting GelDoc photograph can be seen below:

Conclusions