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Contents 1 Lab Work - 21/08/09 1.1 Stochastic Switch team 1.1.1 Summary 1.1.2 Miniprep of C0178 cultures 1.2 Metal Sensing Team 1.2.1 Introduction 1.2.2 Practical Outline 1.2.3 Procedure 1.2.3.1 DNA gel electrophoresis 1.2.3.2 Midi Preps 1.2.3.3 PCR Reactions 1.2.4 Results

Lab Work - 21/08/09

Stochastic Switch team

Summary

Today the stochastic team split into two, to fully utilise the time in the lab. Jess carried out minipreps for the BBa_C0178 in E.coli DH5alpha cultures that were set up yesterday. Goksel carried oud restriction digests of the BioBricks R0062; R0079; C0161; C0179; J44000 which were miniprepped yesterday.

Miniprep of C0178 cultures

As it happens only 2 of the 3 cultures grew from yesterdays overnights so these were spun down (1ml of each). It was decided that these cultures would be treated separately until after being run on a gel so their identities could be confirmed.

After spinning the cultures down after the addition of solution III, it was observed that there was not much of a protein pellet to be seen which should precipitate after solution III is added. The tubes were shaken well and put in to centrifuge for a further 10 miutes and a larger pellet was seen.

N.B: During our minipreps we have always kept solution III on the bench, however Chris seeing this advised us to always keep solution III in the fridge for the best results. As well as this after the addition of solution III the tubes must be shaken well in order for the proteins to precipitate properly. Remember that Phil's protocols may not always be explicit and although care should be taken in following them, there may be important points missed out that can effect results.

The supernatant was taken and Isopropanol added, the solution was spun, ethanol added, aspirated and speed vac'd according to the protocol. The DNA pellet was resuspended in 50ul water, labelled, and frozen in the -20 freezer.

Metal Sensing Team

Introduction

In yesterday's lab session the Metal Sensing team carried out a miniprep of the plasmid pSB1A3, which contained the BioBrick BBa_J33206 (taken from the Parts Registry). This BioBrick contains the arsR binding site as well as the arsR gene - both of which are needed in our system. Once the minipreps were conducted, 10 microlitres of plasmid DNA were taken from three plasmid (pSB1A3) samples (the three samples originating from the three cultures of E.coli containing BBa_J33206 extracted from the plate)and placed into one set of 3 Eppendorf tubes; and then another 10 microlitres were taken from the three samples and put into another set of 3 Eppendorf tubes.

To the first set of 3 Eppendorf tubes, no enzyme was added and to the second tube, a combination of EcoRI and PstI was added. Once the restriction enzyme digests had taken place they were placed in the -20C freezer for use today.

Another thing which was carried out yesterday was preparation of agarose gel; the agarose gel was made up in the usual way, poured in the usual way and when it had set was stored in the fridge (this was done by sprinkling 1 x TAE buffer on the gel surface and then wrapping it in cling film). This gel will be used to analyse the restriction enzyme digests of the pSB1A3 plasmid.

There are still two tasks which need to be resolved:

• Run both the undigested and digested plasmids on agarose gel in DNA gel electrophoresis.
• Conduct midi-preps of the plasmids if gel results are positive.

In addition to this, the Metal Sensing team are hoping to carry out a test PCR reaction on the genome of Bacillus subtilis with the pGFP-rrnb plasmid integrated within it. The Metal Sensing team have designed primers which are embedded within the amyE region of the Bacillus subtilis chromosome so any integration at that site (the pGFP-rrnb plasmid integrates in the amyE gene in B. subtilis) will be detected.

Practical Outline

The tasks needed to be completed by the Metal Sensing team by the end of the day are:

• Run both the undigested and digested plasmids on agarose gel in DNA gel electrophoresis.
• Conduct midi-preps of the plasmids if gel results are positive.
• Run a test PCR reaction on a crude extraction of the genome of the Bacillus subtilis which had successfully been transformed by the plasmid pGFP-rrnb

Procedure

DNA gel electrophoresis

DNA gel electrophoresis was used to analyse the restriction enzyme digests for the three samples of the plasmid pSB1A3 containing BioBrick BBa_J33206. Three samples set aside for electrophoresis contained no restriction enzymes where as three samples of the BioBrick were treated with both EcoRI and PstI. The protocol used to prepare the DNA and to load the samples into the gel can be found at these links.

The way in which the samples were set up in terms of DNA gel wells were as follows:

• Lane 1 = blank
• Lane 2 = DNA ladder (HindIII ladder)
• Lane 3 = Sample 1 (BBa_J33206) + no enzymes
• Lane 4 = Sample 2 (BBa_J33206) + no enzymes
• Lane 5 = Sample 3 (BBa_J33206) + no enzymes
• Lane 6 = blank
• Lane 7 = Sample 1 (BBa_J33206) + EcoRI + PstI
• Lane 8 = Sample 2 (BBa_J33206) + EcoRI + PstI
• Lane 9 = Sample 3 (BBa_J33206) + EcoRI + PstI
• Lane 10 = DNA ladder (HindIII ladder)

The only change to the protocol is that 8 microlitres of ach of the prepared samples were placed into the wells. This is because the pSB1A3 plasmid is a high copy plasmid so the concentration of DNA will be high. Apart from that, the protocol was adhered to.

The results from the gel can be seen in the 'Results' section below.

Midi Preps

PCR Reactions

Results