Construction!

iGEM PROMOTER CHARACTERIZATION

We need to use a medium strength promoter for our constructions, so we characterize different promoters constitutive belonging to the isolated family from Anderson collection (J23101, J23102, J23104, J23107, J23108, J2311, and J23115) these promoters were assemble with GFP to determine promoter strength, using Victor X3 Multilabel Plate Reader.

Result

The characterization of the BioBricks shown in the following table:

 

 

 

 

 

The constructions that were made, were confirmed by double digestions. The following image shows a gel double digestion with XbaI and SpeI for confirmatory promoter constructs:

The constructions were cultivated in LB petri dishes with chloramphenicol and they were observated at UV light after it’s incubation.

In the following graphs there is shown the GFP expression in function of th time and the realtive promoter force.

With the previous results of the characterization of the promoters there is concluded that the promoter J23107, is the strongest because it produces more RPUs.

After characterization, J23104 promoter was chosen for construction of the system of regulation of light and oxygen dependent.