Part:BBa_K4307001

Cro1

Cro protein is a phage-expressed protein, which can relieve the inhibition of cI protein by preventing the synthesis of the repressor.

We first found a combination of cI proteins and Cro proteins reported in a literature, and intercepted the corresponding OR1-OR2 sequences in the literature and applied them to our system. The Cro from the literature is called Cro1.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of Cro1.

Fluorescence assay was done to characterize the biobrick.

To analyze the effect of Cro1 against the inhibitory effect of cI protein on the OR1-OR2 promoter, we integrated Cro1 with the pBAD promoter and placed EGFP fluorescent protein downstream of the OR1-OR2 promoter. After using arabinose to induce the expression of Cro1, the fluorescence results of EGFP protein was compared with the positive control (the bacteria contain plasmid without cI protein nor Cro1 protein) and the negative control (the bacteria contain plasmid with cI protein and Cro1 protein, but Cro1 was not induced). It should be mentioned that cI protein located downstream of its natural promoter (OR2-OR3) and was expressed constitutively in bacteria.

In this experiment, the fluorescence signal of EGFP was recorded at 16th hour after induction with 1% (w/v) arabinose. Besides the fluorescence, the OD600 was measured in order to normalize 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. The OD600 and fluorescence signal was recorded in a plate reader after 16 hours induction at 16°C.

Figure 2: Fluorescence assay for the verification of Cro1.

Conclusion

Cro1 can express normally at 16℃ and can effectively relieve the inhibition of cI protein on the promoter, with a recovery rate of 83.4%. (Recovery rate=(14.76-12.15)/(15.28-12.15)) But Cro1 does not work well at 37℃, either because it is not expressed properly or because the protein forms inclusion bodies in the bacteria.