Difference between revisions of "Part:BBa K1949060"

Revision as of 07:54, 13 October 2016

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 [http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki]). We therefore considered using the improved Prhl (BBa_K1529310,BBa_K1529310) established by Tokyo_Tech 2014, but we noticed that they were inappropriate for two reasons (see[http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki]). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (BBa_K1949060) that suited our goal.

Improvement

Our purpose is to create strong Prhl for our final genetic circuits. This experiment consists of the three parts below.

I. Improved Prhl by Tokyo_Tech 2014 and characterize rhlR assay

II. Improvement of the native Prhl

III. Comparison of the improved Prhl by Tokyo_Tech2014 to our original improved Prhl

The past improved Prhl did not suit for our final circuits and we could create the improved Prhl appropriate to our final circuits.

I. Improved Prhl by Tokyo_Tech 2014 and characterize RhlR assay

We found that Prhl(RL) (BBa_K1529300) activity was weak and the expression level depended on ssrA tag (Fig.1); ssrA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (BBa_K1529310) activity was strong and unexpectedly reacted with C12 (crosstalk) (Fig.4).

Fig. 1. Comparison of the Past improved Prhl and RhlR

Fig. 2. Comparison of the Past improved Prhl and RhlR adding high concentration C4

The colonies of transformants with a rhlR (BBa_C0171) plasmid looked rough and the growth rate was low(Fig.2-left), 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. 3. The colonies of transformants with a rhlR (left) or a rhlR-ssrA（right)

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 activity was stronger than native Prhl(Fig.4).

Fig. 4. RFU of GFP / turbidity of imoroved Prhl mutants

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

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

Fig. 5. Comparison of Prhl(NM) to Prhl(LR)

Sequence and Features