Composite

Part:BBa_K1507013

Designed by: Willy Hsu   Group: iGEM14_NTU_Taida   (2014-10-07)

Fatty acid induced expression auto regulator (J23119-B0034-FadR-B0015- J23119-B0034-FadL-B0015)

A down-counting timer system. The expression of GFP (green fluorescence protein) will be shut down after a period of time whenever the bacteria uptakes fatty acid.

Testing Result: Function of J23119-RBS34-FadR-terminator-J23119-RBS34-FadL-terminator

Purpose:

Confirm that FadR protein and FadL protein assist the bacterial uptake of fatty acid.

Method:

1. We use modified LB medium for culturing E.coli strain. The amount of yeast extract is decreased.

tryptone 10g

yeast extract 1g

NaCl 10g /1L ddH2O

We added 20λ, 40λ, 60λ, 80λ, 100λ oleic acid in 2mL modified LB medium respectively. This fatty acid – composing broth is used for culturing E.coli.

2. The E.coli strain with FadR/FadL coding gene and the DH5α cell (without FadR/FadL coding gene) is cultured overnight in the modified LB medium, 2 c.c respectively, with different fatty acid ratio.

3. Record the absorbance of the suspension culture by spectrophotometer.

Since the amount of yeast extract is decreased significantly in LB medium, the bacteria is forced to metabolizes oleic acid for carbon source (yeast extract is the main carbon source in origin LB medium). Therefore, the E.coli strain that contain FadR/FadL coding gene will be more adjustable to the critical environment and grow better in the medium. We compared the growing condition of FadR/FadL (+) and FadR/FadL (-) strain in modified medium.

Result:

% of oleic acid plasmid (-) plasmid(+)
1% 2.542 2.661
2% 2.390 2.623
3% 2.523 2.357
4% 2.610 2.616
5% 2.351 2.653

Kexp2.jpg

FadR/FadL(+) E.coli strain grows better in the critical environment, compared to the control group. The phenomenon suggests FadR and FadL protein play important roles in the uptake and metabolism of fatty acid in E.coli. Fatty acid sensing system can be designed on the basis of the dynamic interaction between FadR-acyl-coA complex , fatty acid molecules and pfadBA promoter

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 916
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 927
    Illegal NheI site found at 950
    Illegal SpeI site found at 916
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 916
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 916
    Illegal AgeI site found at 2081
  • 1000
    COMPATIBLE WITH RFC[1000]


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