Difference between revisions of "Part:BBa K817001"

Revision as of 02:03, 6 October 2012

FadR

It's a construct encoding natural repressor for pfad, a natural fatty acid sensing promoter.

Method

To evaluate the P_fadBA promoter function, we design a P_fadBA-mRFP reporter construct. In experimental group we add oleic acid in media and compared to control group, which is the same colony and identical amount of bacteria but without addition of oleic acid. After 5 hr induction we measure the man fluorescence intensity to get the expression level of P_fadBA promoter in either group.

However, the baseline expression of the reportor gene (mRFP) downstream of PfadBA is so high that we cannot see significant signal rise after induction of fatty acid (oleic acid). Therefore, we decide to lower the baseline expression by overexpression of FadR, an endogenous repressor of PfadBA, whose repressive function is antagonized by fatty acyl-CoA. [3] By co-transforming constructs with PfadBA and FadR into the bacterial platform, it is made capable of changing gene expression in response to environmental fatty acid concentration.

Protocol

1. 10 μL bacterial culture was cultured in 5 mL LB at 37。C, with suitable concentration of antibiotics shaking for 18 hr.
2. For experimental group, 480 μL of bacteria culture was transferred to 1.5 mL eppendorf tube and added with 20 μL of oleic acid and IGEPAL(detergent) in. For control group, 500 μL bacterial culture was added in 1.5 mL eppendorf tube.
3. The tubes were incubated at 37℃ 5 hr for induction.
4. 100 μL of bacterial culture was transferred tino 96-well plate. The mRFP fluorescence intensity was measured (Excitaion: 580 nm, Emmision: 610 nm).

Data

Oleic acid induction group shows higher expression of mRFP.

Conclusion

Our P_fadBA promoter can work in response to environmental fatty acid change, which acts as an important sensor for our bacterial device – secreting GLP-1 when host is fed with fatty diet.

Sequence and Features