From 2014.igem.org

• HOME
• PROJECT
  • Overview
  • Backgroud
  • Production module
  • Pumping out module
  • Indicator module
• ACHIEVEMENT
  • Parts test results
  • Modeling
  • Judging Criteria
• WET LAB
  • Protocol
  • Notebook
  • Parts
  • Cooperation
• HUMAN PRACTICE
• SAFETY
• TEAM
  • Member
  • Attribution
• ACKNOWLEDGEMENT

Indicator module

In this module, we try to figure out a method to monitor the fatty acids producing status. Specifically, not only could our E.coil automatically “inform” us of when fatty acids have been produced greatly, but it could also “show” us the relative concentration of fatty acids compared to control E.coli. To fulfill this objective, we need to find out something sensitive to fatty acids amounts in E.coli. Thus, we noticed the native regulatory system in E.coli fatty acids metabolism..

Working principle

In E.coli, the fad genes family is responsible for fatty acid degradation while the genes family fab works in the synthetic pathway. The protein FadR is a regulator. Generally, it binds to the promoters of fadD and fabB, and works as repressor and activator, respectively. In the absence of long chain acyl-CoA, FadR will repress the fad regulon, activating the transcription of fabB and starting the fatty acid synthesis. Otherwise, the binding of long chain acyl-CoA to FadR will dramatically change the structure of this protein and FadR will be released from the operator site. Then the fad regulon transcription increases whereas that of fabB decreases. In this case, redundant fatty acids can be degraded for other usage.

Our circuit

Based on this, we built an indicator structure similar to traffic light by introducing the gfp and rfp genes. As is known, pfadD and pfabB were the promoters of fadD and fabB which we isolated from wide-type E.coli. After assembling them with two fluorescent genes at their downstream(as shown in the figure below), they could regulate the transcription of rfp and gfp through the general concentration of fatty acid.

When high concentration of fatty acid have been produced, which means long chain acyl-CoA could be produced, FadR would dissociate from the binding site and the “red light" would be turned on (RFP would be expressed). In contrast, in the situation that concentration of fatty acid is low, there isn’t analog competing to bind to the operator site. Hence FadR tightly binds to the operator site and the green light will be turned on (with GFP expression).

Reference

1. Yasutaro F, Hiroshi M, Kazutake H: Regulation of fatty acid metabolism in bacteria. Molecular Microbiology 2007, 66(4):829–839

2. Liu H, Yu C, Feng D, Cheng T, Meng X, Liu W, Zou H, Xian M: Production of extracellular fatty acid using engineered Escherichia coli. Microbial Cell Factories 2012, 11:41-53

3. Yasutaro F, Hiroshi M, Kazutake H: Regulation of fatty acid metabolism in bacteria. Molecular Microbiology 2007, 66(4):829–839

4. Feng Y, Cronan JE: Crosstalk of Escherichia coli FadR with Global Regulators in Expression of Fatty Acid Transport Genes. PLOS ONE 7(9): e46275