Part:BBa_K4307034
cI (lambda)
cI protein is a repressor expressed in bacteriophage. It binds to part of the right operator (OR) of phage, which has the promoter feature (OR1+OR2), and inhibits the binding of RNA polymerase to the promoter region.
Naturally, the expression of cI protein is regulated by another part containing promoter feature in the right operator (OR) of the phage, which can interact with Cro protein and subsequently lose its promoter function. Thus, the expression of cI protein is regulated by Cro proteins.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The following figure demonstrates our successful construction.
Fluorescence assay was done to characterize the biobrick.
In order to demonstrate the inhibition efficiency of cI protein on the OR1-OR2 promoter, we use EGFP as the reporter gene to reflect the function of OR1-OR2. The negative control is the bacteria contain an empty plasmid, and the positive control is the bacteria contain plasmid with OR1-OR2-EGFP and cI protein. It should be mentioned that cI protein is located downstream of its natural promoter (OR2-OR3) and expressed constitutively in the strain.
The fluorescence signal was recorded 16 hours after inhibition at 37°C or 16°C. OD600 was measured in order to do standardization. In the following figure, those values represent the average result of two repeated normalised results.
Conclusion
Compared with the negative control and positive control, it can be claimed that the inhibition effect of cI protein on the OR1-OR2 promoter is very strong, since the expression of downstream genes can be inhibited almost completely. This give us more confidence to build logical gate.
| None |