We have successfully cloned SpPCS in pSB1C3 (BBa_K1526000) into E.coli DH5α
We have successfully cloned the intermediate construct pYodA + SpPCS in pSB1C3 (BBa_K1526004) into E.coli DH5α
We have successfully cloned Gsh1* (BBa_K1526001) in pSB1C3 into E.coli DH5α
We have successfully cloned CysP in pSB1C3 (BBa_K1526003) into E.coli DH5α

Characterizing the parts:

In order to characterize the parts we have created a new BioBrick compatible with our chassis. As E.coli DH5α does not have a lac repressor system we have assembled together basic parts J32100, B0033, C0012, B0010, B0012 and R0010 to create the composite regulator Lac Repressor System (LacRS) Our first functionalization test coupling LacRS upstream the Green Fluorescent Protein (GFP) Biobrick (E0040) showed expression of GFP under UV light when the system is previously induced by IPTG.

We have successfully cloned and tested the functionality of LacRS in pSB1A3 (BBa_K1526009) into E.coli DH5α

Creating Inducible monofunctional constructs:

Each gene from our project was assembled downstream our LacRS Biobrick in order to allow a better characterization of the parts.

We have successfully cloned LacRS+Gsh1* in pSB1C3 (BBa_K1526005) into E.coli DH5α
We have successfully cloned LacRS+SpPCS in pSB1C3 (BBa_K1526006) into E.coli DH5α
We have successfully cloned LacRS+MntH (BBa_K1526007) in pSB1C3 into E.coli DH5α
We have successfully cloned LacRS+CysP in pSB1C3 (BBa_K1526008) into E.coli DH5α

Characterising CysP:

In order to characterise the sulfate secondary transporter from Bacillus subtilis we have adquired E. coli BW25113 strain mutants from the Keio collection for complementation assays.

We have used CysW (GenoBase code: JW2416) and CysA (GenoBase code: JW2415) mutants from the Keio collection as they represent deletions of different subunits (respectively, permease and ATPase) of the main sulfate transporter in E. coli (ABC transporter).

We have transformed both the mutants and the wild type with our LacRS+CysP plasmid and put them to grow on M9 media (1mM of Sulfate and 0.4%w/v of glucose) .

We still did not have time to make the optical density measurements on a 96-well plate on the FlouStar Omega plate reader kindly loaned to our team by BMG.