Formal Lab Session - 16th September 2009

Stochastic Switch Team

Today we ran the ara PCRs from yesterday on a gel and got a really good result, this product was then cleaned, cut with EcoRI and SpeI, cleaned again, then ligated with the pre-cut pSB1AT3 plasmid backbone.

Today we also did minipreps of 12 each of the sac and ara cultures... however unfortunately today the centrifuge we had been relying on broke, and therefore all three teams were forced to use one small centrifuge. This meant that the minipreps could end up being of poor quality due to waiting times for the centrifuge.

Metal Sensing Team

Introduction

In the last lab session, the Metal Sensing picked 12 colonies from a plate containing E. coli + cotC-GFP-smtA transformants and used them to inoculate 12 tubes containing LB + ampicillin - these wil be used for mini-preps today. In yesterday's session we also discovered that the attempts to transform E. coli with the arsR-cadA cadmium sensor failed, so on that same day we carried out a second attempt at transforming E. coli with this BioBrick.

Today's lab session will be focussed on carrying out the mini-preps for the 12 colonies of potential E. coli + cotC-GFP-smtA transformants and based on analysis, will determine whether midi-preps will follow or whether we need to reattempt another transformation (we would, of course, have to carry out another ligation reaction of the cotC BioBrick and the pMUTIN4 plasmid first)

To prepare for the eventuality that the ligations, transformations and midi-preps of the cotC-GFP-smtA in pMUTIN4 worked, the "Evening before transformations day" steps of the Bacillus subtilis transformation protocol will be carried out too.

The plate containing the cadmium-sensor E. coli transformants will also be studied - if there are colonies, 12 of them will be prepared for mini-preps; if there is no success then ligation of the BBa_J33206 BioBrick (promoter removed) with the cadA promoter will be carried out - E. coli cells will then be transformed with this.