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Lab Work - 09/09/09

Metal Sensing Team

Introduction

In the last lab session the Metal Sensing Team had shown that the version of the BBa_J33206 BioBrick submitted by Chris French (contained within the pSB1A2 plasmid) was indeed the correct one. However any further work on this BioBrick should now be put on temporary hold because of the arrival of our newly synthesized BioBricks. These synthesized BioBricks need to be transformed in E. coli bacteria as well as other processing measures.

Today's task is to tranform DH5-alpha E.coli bacteria with our newly synthesized BioBricks - one set of cells should be tranformed with the cotC-GFP-smtA BioBrick and the other group of cells should be transformed with the kinA BioBrick

Procedure

To carry out the transformations of the two sets of DH5-alpha E.coli cells with our two BioBricks, we adhered to Dr. Aldridge's transformation by heat shock protocol. The only differences to the protocol were these:

• Step 2 - the period in which the DH5-alpha cells were left on ice was 15 minutes instead of the indicated 30 minutes.

• Step 4 - the amount of DNA added to the DH5-alpha cells (whether it was cotC-GFP-smtA BioBrick DNA or kinA BioBrick DNA) was 10ul.

• Step 5 - the cells were left on ice for 15 minutes instead of the suggested 3 minutes.

Once the transformation procedure for both sets of DH5-alpha E.coli cells was completed they were plated out on some LB+kanamycin plates (seen as kanamycin is the antibiotic the plasmid pMK-RQ offers resistance to). The way in which the transformants were plated out were as follows:

• 200ul of the cotC-GFP-smtA transformed E. coli cells were plated on an LB + kanamycin plate
• 200ul of the kinA transformed E. coli cells were plated on an LB + kanamycin plate

The remainder of the two sets of cells were then centrifuged for 1 minute. 500ul of the supernatant was then removed and pellet resuspended in the remaining solution. The resuspended transformant cells were then placed on plates as follows:

• 500ul of the resuspended cotC-GFP-smtA transformed E. coli cells were plated on an LB + kanamycin plate
• 500ul of the resuspended kinA transformed E. coli cells were plated on an LB + kanamycin plate