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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

The midi-preps were conducted, as usual, using the Sigma-Aldrich Midi-prep kit and protocol. There were no changes to the protocol and 10ul samples of the five midi-prepped plasmids were loaded onto 0.8% agarose gel (see the 'Results' section below). The midi-prepped DNA samples were also assessed for DNA concentration using the NanoDrop spectrophotometer.

cotC-GFP-smtA

The following diagram shows the concentration of the cotC midi-prep sample as well as the DNA to Protein ratios; at 89.7 ng/ul the DNA concentration is at a suitable level for further processing. When analysed through DNA gel electrophoresis (see 'Results' section below) it can be seen that the band produced by the cotC-GFP-smtA within the uncut pMK-RQ plasmid lies just below the 2027bp fragment of the HindIII ladder.

Whilst the plasmid + BioBrick is 3,663bp the plasmid was uncut so it appears as a shorter fragment in the gel. To prove that the midi-prepped DNA is 3,663bp, we should carry out restriction enzyme digests on another day.

kinA

The following diagram shows the concentration of the kinA midi-prep sample as well as the DNA to Protein ratios; at 20.8 ng/ul the DNA concentration is not as good as expected but still enough for further processing. When analysed through DNA gel electrophoresis (see 'Results' section below) it can be seen that the the kinA + pMK-RQ plasmid (uncut) band lies between the 2027bp and 2322bp fragment of the HindIII ladder.

Whilst the plasmid + BioBrick is 4,327bp the plasmid was uncut so it appears as a shorter fragment in the gel. Likewise, to prove that the midi-prepped DNA is 4,327bp, we should carry out restriction enzyme digests on another day.

pGFP-rrnB

pMUTIN4

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:

The lanes are as follows:
Row 1

• Lane 1 = blank
• Lane 2 = HindIII DNA Ladder
• Lane 3 = cotC midi-prep sample
• Lane 4 = kinA midi-prep sample
• Lane 5 = pGFP-rrnB midi-prep sample
• Lane 6 = pMUTIN4 midi-prep sample
• Lane 7 = pSB1AT3 midi-prep sample
• Lane 8 = blank
• Lane 9 = cotC PCR reaction 1 sample
• Lane 10 = cotC PCR reaction 2 sample
• Lane 11 = cotC PCR reaction 3 sample
• Lane 12 = cotC PCR reaction 4 sample
• Lane 13 = blank
• Lane 14 = pMUTIN4 midi-prep sample (from second attempt)
• Lane 15 = leak-over of sample from lane 14
• Lane 16 = pMUTIN4 midi-prep sample (from second attempt)
• Lane 17 = HindIII DNA Ladder

Row 2

• Lane 1 = blank
• Lane 2 = HindIII DNA Ladder
• Lane 3 = sac midi-prep digest sample
• Lane 4 = ara midi-prep digest sample
• Lane 5 = sleB midi-prep sample
• Lane 6 = HindIII DNA ladder

Conclusions