Team:Newcastle/Labwork/19 August 2009

=Formal Lab Session - 19th August 2009= = Overview = 
 * Metal Sensor Team - picked 3 colonies of potential BBa_J332066 E. coli transformants and inoculated LB solution with them
 * Stochastic Switch Team - only 5 out of 7 BioBricks had transformed in E. coli cells - these colonies were used to inoculate LB solution
 * Sporulation Tuning/Chassis Team - counted the colonies produced by the treated sleB/cwlJ mutants

Introduction
So far in the lab, we have prepared the BioBrick BBa_J33206 (arsR promoter + coding sequence) from the repository and attempted transformation of this DNA in E.coli. Once this procedure had been completed, the Metal Sensing team then streaked the bacteria on an LB + ampicillin plate and left it on 37°C incubation overnight.

Today’s procedure involves searching the plate for any colonies and if any colonies are present, picking three of them and inoculating 3ml LB with them. These grown up cultures will then be used for a mini-prep of the BBa_J33206 BioBrick. If no colonies are present, we must attempt transformation again. Because the plasmid involved in this BioBrick (pSB1A2) is a high copy plasmid, we can inoculate 3ml LB + ampicillin in the morning; by the afternoon the E.coli should have grown in the LB solution sufficiently to further inoculate 50ml LB solution in the afternoon (for midi-preps).

Practical Outline
These are the steps that the Metal Sensing Team want to have completed by the end of the day:
 * 1) Innoculate three tubes (each with 3ml of LB) with three of the E.coli cultures containing BBa_J33206 - morning
 * 2) Start preparing for a test PCR for the pGFP-rrnb plasmid in Bacillus subtilis.
 * 3) Should the E.coli cultures (containing BBa_J33206) have grown sufficiently in the LB tubes, further inoculate 3 flasks (containing 50ml of LB) with these bacteria – afternoon.

Observations and Procedure
When looking at the plate following an overnight incubation it could be seen that the transformation rate for the DH5α E.coli containing BBa_J33206 DNA was very low – only five colonies were spotted on the plate. Nevertheless three colonies were removed from the plate and, under aseptic conditions, used to inoculate 3 tubes containing 3ml LB each. These tubes were then placed in the shaking incubator at 37°C for the rest of the day. This step was done first thing in the morning. The bacteria which grow in these LB tubes will be used for a mini-prep of the BBa_J33206 BioBrick tomorrow

In the afternoon, the team returned to the lab to observe the progress of the E.coli bacterial growth in the three LB tubes. The appearance of the three tubes was noted as cloudy which indicated the presence of sufficient numbers of E.coli bacteria. To save time in the labs, it was decided to inoculate 3 flasks (each containing 50ml LB) with these three samples – these 50ml flasks will then be used for midi-preps tomorrow should the mini-preps indicate that the E.coli have been successfully transformed.

To inoculate the 3 flasks containing 50ml LB each, the team took 0.5ml from each of the 3ml of cultures and pipette them into the separate flasks. These flasks were then placed into the orbital shaking incubator at 37°C overnight.

Summary
Today we discovered that only 5 of 7 of our bricks had been transformed. Biobricks C0178 and C0062 had not worked. We decided to plate out 400ul of the remaining culture that we had left from yesterday onto a appropriate plates for both of these bricks in hope of getting some growth for miniprep. Today we also set up 3ml overnights for the remaining 5 bricks that worked (R0062; R0079; C0161; C0179; J44000). We did these in plain LB, later realising that we should maintain the selection for the Amp resistance plasmid by setting up the overnights in LB+antibiotic.

Summary
Today, we came in to check out the plates for the sleB/cwlJ mutants which we treated [yesterday].

In order to obtain accurate results, we counted the colonies, as can be seen in the following pictures.



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