Week 1

This week we transformed DNA from BBA_J04450 (RFP construct) in plasmid backbone pSB1C3 to test how efficient our competent cells were.

Week 2

In week two we transformed E. coli with DNA from BBA_K592009 amilcp.

Week 3

Our team were at YSB for the majority of this week therefore no lab work took place.

Week 4

We transformed E. coli with pSB1K3 and pSB1A3 so we were able to again test how efficient our competent cells were as we had had to make up more competent cells.

Week 5

We transformed parts from the kit plates for constructing the lac repressor system (see description below), and also those necessary for testing our pYoda promoter into DH5α. We also carried out the first assembly step for making the system.

In order to characterise our parts we needed to be able to induce expression to show that our genes only have an effect when expression is induced. For this we constructed the lac repressor system:

The gene to be characterised would be ligated downstream, and expression induced using IPTG. This works as follows: J23100 is a constitutive promoter, resulting in constitutive expression of the LacI repressor protein, which represses expression of the target gene. IPTG can be added to inhibit LacI, thus inducing expression of the target gene.

The system is built from the following parts available in the 2014 distribution:

BBa_J3100	BBa_P0312	BBa_R0010
Constitutive Promoter	Encodes LacI	Promoter inhibited by LacI

Week 6

We gel extracted digested backbones en masse. The 2nd assembly stage of the Lac repressor system carried out, and we gel checked numerous colonies. Our synthesised genes finally arrived (in pUC57), so we could start work with them. The first step was to transform the plasmids into DH5α.

Week 7

To further check that our lacI repressor system is the correct construct, we ligated it with GFP as the test gene. When induced with IPTG, colonies containing this construct were visibly green in normal light. Work on the main project continued as outlined below:

1. Our synthesised genes were delivered in pUC57. We transformed E. coli with the genes in pUC57, and carried out miniprep to increase the quantity of DNA we had available to work with.

2. The genes of interest were amplified by PCR using the primers BioBrick-f and BioBrick-r which anneal to the prefix and suffix of the biobricks. (After previous problems with 3A assembly, we decided to amplify our genes by PCR before digesting them to ensure there were no other backbones present, in case these were ligating with the desired backbone rather than the parts themselves.)

3. Digestions were carried out:

   • Each part digested with EcoRI and PstI for cloning into pSB1C3 to send to the registry.

   • For for combining with each other in pairs, as shown in the scheme below to work towards the final construct.

   Digested DNA was ligated together and transformed using chemocompetent cells.

4. Gene sizes were checked by colony PCR.

   Scheme For Construction of Final Construct

   
   
   

5. Characterisation of Parts

Characterisation of pYoda is taking place using GFP as a reporter. To test how inducible to Cadmium pYoda is, Cells are exposed to different concentrations of Cadmium, and fluorescence is measured over a time course. Fluoresence is measured relative to that of GFP under the control of the constitutive promoter BBa_I20260, and in pSB3K3, to enable conversion to absolute units.
CysP. CysP from Bacillus subtilis is a sulfate permease, [orthologous/homologous/some way similar] to [insert mutant genes] in E. coli. Thus we are characterising CysP by transforming it into cysA and cysW mutants and measuring the effect on their growth.