Difference between revisions of "Part:BBa K1846005"

Revision as of 05:37, 20 September 2015

cI-Cro bacteriophage lambda lytic cycle regulatory circuit

The cI-Cro construct contains a circuit for the production - via a constitutive promoter - of the cI repressor protein (also known as the Lambda Repressor), which is responsible for keeping bacteriophage lambda in the lysogenic cycle through the cooperative binding of two repressor dimers to the DNA, repressing the Cro gene. The circuit uses a T7 promoter to drive the expression of the Cro gene in the opposite direction to the cI gene, essentially silencing the expression of the cI gene. The strength of the promoter used means that in the presence of T7 DNA polymerase, production of the Cro repressor protein will incapacitate the production of the cI repressor protein. Thus, the presence of the T7 RNA polymerase in the system enables the switch from the lysogenic cycle to the lytic cycle.

We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 3). As cI repressor protein is produced constitutively in our circuit, we expected to see a cI repressor protein band at ca 26 kDa (cI repressor protein = 237 aa, MW: 26211.8 Da). Our control E.coli 10β cells containing the cI/Cro construct are showing the expected bands at ca 26 kDa (Figure 3, samples 7, 8 and 9). We attribute the lack of cI protein bands in our T7 Express cell lysate (Figure 3, sample 4) to T7 RNAP leakage, which would silence the expression of the cI repressor protein through the expression of Cro repressor protein in the opposite direction. Absence of Cro repressor protein bands (Cro repressor protein = 66 aa; MW: 7363.4 Da) could be due to protein overexpression and aggregation into insoluble fraction (due to the strength of the T7 promoter). This hypothesis requires further investigation, however our preliminary results suggest the circuit would be functional.

http://2015.igem.org/File:BBKiGEM-SDSgel-Figure_3.jpg

Sequence and Features