Difference between revisions of "Part:BBa K4307044"

Latest revision as of 15:19, 13 October 2022

OR2-OR3-cI + pBAD-cro

cI/Cro is a natural feedback system in bacteriophages, which acts as a bidirectional switch to determine the fate of bacteriophages and the infected bacteria. Phage genome has two operators, called OR (the right operator) and OL (the left operator). OR plays an important role in the regulation of gene expression of phage. The cI protein is a phage-encoded repressor that binds to the OR. After binding to OR, cI protein will exclude RNA polymerase from binding to PR (located within OR) and inhibit the expression of the downstream gene. Cro protein is another phage-expressed protein, which can relieve the inhibition by preventing the synthesis of the repressor.

Considering that the phage genome can be integrated into the bacterial genome in lysogenic bacteria, we isolated the the part related to the expression of cI (OR2-OR3) in OR and integrated it into the bacterial genome together with the downstream cI gene. The Cro gene in phage was isolated and integrated with pBAD promoter. Thus, we successfully reproduced the bidirectional switch in bacteriophage using OR2-OR3-CI and PBAD-CRO in E. coli.

Sequence and Features

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of OR2-OR3-cI + pBAD-cro.

Figure 2: The construction results of OR2-OR3-cI + pBAD-cro.

Fluorescence assay was done to characterize the biobrick.

To ensure the normal expression of cI and Cro, we added the gene of EGFP downstream of the promoter (OR1-OR2) that can be inhibited by cI protein to examine whether the isolated promoter (OR2-OR3) can effectively promote the expression of cI, and whether pBAD can successfully induce the expression of Cro with low leakage. We tried two homologous Cro proteins with different sequences but identical matched cI protein.

In this experiment, the fluorescence signal of EGFP (Ex. 485 nm, Em. 520 nm) was recorded at 16th hour after the induction with 1% (w/v) arabinose. Apart from the fluorescence, the OD600 was measured in order to standardize the fluorescence signal per cell. All groups were carried out twice to do a statistical analysis. Different experiments were induced in one 96 well plate. In order to exclude the interference of impurities, we use PBS to resuspend the bacteria before the measurement of OD600 and fluorescence signal. The OD600 and fluorescence signal was recorded in a plate reader after 16 hours induction at 16°C, while the OD600 and fluorescence signal of Cro2 protein was recorded in a plate reader after 16 hours induction at 37°C or 16°C.

Figure 3: Characterization of cro/cI function by fluorescence spectrophotometry.

Conclusion

Through the above verification, we proved that Cro and cI can also play similar regulatory roles on bacterial plasmids as in phage genomes, cI protein has a good inhibitory effect, and Cro2 has a better disinhibitory effect on cI protein than Cro1. The cI/Cro system has the potential to be used as a bidirectional switch or logic gate system in bacteria, which is our contribution to the iGEM community.