Part:BBa_C0171

rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)

same as C0071 except no LVA tag

Characterization

Group: Tokyo Tech 2016

Author: Yoshio Takata

We simulated our final genetic circuits and found that the circuits did not work, because Prhl activity was too weak compared to Plux. (see the Modeling page and the Only Assay page). 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 the Discussion). Then, we decided to improve Prhl Promoter and obtain our original improved Prhl (Noticeable Mutant) (BBa_K1949060), hereafter referred to as Prhl(NM), that suited our goal.

During improving Prhl(R0071), we characterize this part. So, we describe that characterization.

We found that Prhl(RL) (BBa_K1529300) activity was weak and the expression level depended on LVA tag (Fig.1); LVA-tagged proteins are prone to be degraded by cellular proteases. Prhl(LR) (BBa_K1529310) activity was strong and unexpectedly reacted with C12 (crosstalk) (Fig.3).

Fig.1 Comparison of the Past improved Prhl and RhlR

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-LVA (BBa_C0071) plasmid looked smooth and the growth rate was normal(Fig.2-right). However, the reason for this result is unclear.

Fig.2 The colonies of transformants with a rhlR (left) or a rhlR-LVA (right)

If you want more information, you see [http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki] and BBa_K1949060!

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 240
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 715