Part:BBa_K2230006

Pcar-RBS-RFP-T/pSB1A3

Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.

Cloning

Pcar-RBS-RFP was amplified from RFP Coding Device (BBa_J04450) using a primer with Pcar-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.

Function

RFP can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.

Demonstration: Glucose responsive promoter activity

The result shown in Fig. 1 indicated that PI promoter has significant activity in LB culture media. However, the activity of Pcar promoter is greater than negative control but much smaller than PI and positive control. It’s consistent with the properties of PI and Pcar promoters just mentioned previously and described previously in Part Resgistry (BBa_K861170) by team [http://2012.igem.org/Team:WHU-China WHU-China] in 2012 who designed the promoters.

In our experiment as presented in Fig. 2, PI and Pcar promoters just responded to various concentrations of glucose with a very slight dose-dependent increase. This phenomenon didn’t correspond to the data provided by team WHU-China in 2012 and team NCKU_Tainan in 2016. Maybe our measurement was not in an optimized condition. Or the reporter of RFP activity was not sensitive enough to respond this difference.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 617
  Illegal AgeI site found at 729
  
• 1000
  COMPATIBLE WITH RFC[1000]