Part:BBa_K1949060

Prhl(NM)-rbs-gfp

We simulated our final genetic circuits and found that the circuits did not work, because Prhl strength was too weak. (see our wikiリンク貼る). We therefore considered using the improved Prhl (BBa_K1529310, BBa_K1529300) established by Tokyo_Tech 2014, but we noticed that they were inappropriate for two reasons (see our wiki　). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (BBa_K1949060) that suited our goal.

Improvement

I. Improved Prhl by Tokyo_Tech 2014 and RhlR assay We found that Prhl(RL) (BBa_K1529300) strength was weak and the expression level depended on ssrA tag (Fig.1 (a), (b)); ssrA-tagged proteins are prone to be degraded by cellular proteases.

待ち！ Fig.1

The colonies of transformants with a rhlR (BBa_ C0171) plasmid looked rough and the growth rate was low(Fig.2left), while the colonies of transformants with a rhlR-ssrA (BBa_C0071) plasmid looked smooth and the growth rate was normal(Fig.2-right). However, the reason for this result is unclear.

Fig.2

II. Improvement the native Prhl By introducing a single point mutation into native Prhl (BBa_R0071) by PCR, we obtained 198 Prhl mutants and chose the two Prhl mutants of which promoter strength was stronger than native Prhl (Fig.3　※この図は差し替えます). The sequences of these tow chosen mutants are shown in below. The small characters indicate the scar sequence and a single point mutation is colored with red.

Native Prhl (BBa_R0071) TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAAAAAGTGTTCtactagagAAAGAGGAGAAA

Prhl(D6) (BBa_K1949060) TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAtAAAGTGTTCtactagagAAAGAGGAGAAA

Prhl(H7) TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAtAAAGTGTTCtactagtaAAAGAGGAGAAA

Fig.3

III. Comparison the improved Prhl by Tokyo_Tech 2014 to our original improved Prhl

Prhl(D6) was chosen from the many Prhl mutants, and by comparing Prhl(D6) to Prhl(LR), we obtained the result below (Fig.3-2-2-3-4). The reaction strength of Prhl(D6) to C4 was higher than that of Prhl(LR), and Prhl(D6) did not react with C12 at all.

Fig.4