Part:BBa_K4307002
Cro2
The Cro protein encoded by the bacteriophage acts as an inhibitor in the synthesis of cI protein. This inhibition can free OR1-OR2 from its inhibitor, cI protein, thereby helping the express of gene downstream of OR1-OR2.
We first found a combination of cI proteins and Cro proteins reported in a literature. After checking parts bank of iGEM community, we found that HUST (Huazhong University of Science and Technology) has used cI/Cro system. The cI protein they used has the same sequence as ours, but the sequence of Cro protein is different. Therefore, we tried the Cro protein from HUST without replacing cI. (The Cro protein is called Cro2 afterwards.)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 63
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The following figure demonstrates our successful construction.
Figure 1: The construction results of Cro2.
Fluorescence assay was done to characterize the biobrick.
To test whether Cro2 protein can protect OR1-OR2 from being inhibited by cI protein, we used pBAD promoter to control the inducible expression of Cro2 protein, and placed EGFP fluorescent protein downstream of the OR1-OR2 promoter as the reporter signal. Subsequent to utilizing arabinose to induce the expression of Cro2 protein, the fluorescence consequence of EGFP protein was contrasted with the positive control (The E. coli stain contains plasmid without cI protein nor Cro2 protein) and the negative control (The E.coli stain contains plasmid with cI protein and Cro2 protein, yet Cro2 was not induced). It ought to be referenced that the expression of cI protein was constitutively in the negative control and the experiment group (column 2 and column 3 in each culture temperature in figure 1), since cI protein was controlled by its natural promoter (OR2-OR3).
Experiments were performed in E. coli BL21(DE3) cell strain cultured at 37°C or 16°C. After E.coli was induced with 1% (w/v) arabinose for 16 hours, PBS buffer was used to resuspend the bacteria to exclude the interference of impurities. The fluorescence signal of EGFP and the OD600 were measured. OD600 was estimated to standardize the fluorescence signal per cell. All groups were completed two times to get average results. Various samples were placed in one 96 well plate. The OD600 and fluorescence signal was measured in a plate reader following 16 hours induction at 37°C or 16°C.
Figure 2: Fluorescence assay for the verification of Cro2
Conclusion
Cro2 can express normally at 16℃ and can effectively relieve the inhibition of cI protein on the promoter, with a recovery rate of 34.9%. (Recovery rate=(1.50-1.20)/(2.06-1.20)) The recovery rate of Cro2 was lower than that of Cro1 at 16℃. Apart from that, it seems that Cro2 works well at 37 ° C, with a recovery rate of 40.5%. (Recovery rate=(1.10-0.76)/(1.60-0.76))
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