Generator

Part:BBa_K1472606

Designed by: Kai Kin Chi,So Lok Man,Li Lok Yee,Tse Man Chak   Group: iGEM14_CityU_HK   (2014-09-19)

TetR repressible FadL & FadD generator

The part contains a TetR repressible promoter that is upstream of the fadL and fadD genes that are both preceded with a strong ribosome binding site (RBS). It is inducible by anhydrotetracyclines (aTc) to express the two integral membrane proteins FadD and FadL. The fadL gene codes for long-chain fatty acid (LCFA) transporter which can take in LCFA through the outer membrane. Acyl-CoA synthase encoded by the fadD gene further activates LCFA into long-chain fatty acyl-CoA and transports them through the inner membrane into the cytosol (Figure 1). Overexpressing fadD and fadL renders E. coli more efficient in the uptake of LCFA which provides an extra supply of LCFA for its metabolism (via β-oxidation) or other biosynthetic purposes (membrane biosynthesis). This protein generator was used by CityU_HK 2014 team to increase the LCFA uptake for alpha-linolenic acid production in E. coli.


CityU_HK_fad_newbb.jpg


Figure 1. Transportation of long-chain fatty acids facilitated by the integral membrane proteins, FadL and FadD. Adapted from Fujita et al. (2007)


Results and characterization

In an attempt to increase fatty acid uptake in E. coli, we constructed a tetracycline-repressible fadL-fadD expression system (BBa_K142606). As we were aiming to ensure significant co-expression of the fadL-fadD genes in E. coli, we subcloned the fadL-fadD gene cassette to the IPTG-inducible pSNAP tag (T7)-2 expression vector to test the effect of FadL-FadD overexpression on oleic acid uptake. Expression of the fadL and fadD genes in recombinant and control E. coli host cells was measured by qRT-PCR assays, and fatty acid uptake measured by GC-MS analysis.

Quantitative real-time PCR analyses showed that fadL and fadD mRNAs were increased in recombinant fadL-fadD E. coli as compared to the control cells in the absence and presence of IPTG induction (Figures 1A and 1B). For the fatty acid uptake analysis, oleic acid concentration in recombinant and controls cells was measured by GC-MS. Although no difference in oleic acid uptake was observed in the fadL-fadD recombinant and control cells at 0 µM and 40 µM of IPTG, the oleic acid concentration in the fadL-fadD transformant was 2-fold higher at 400 µM IPTG than the control cells (Figure 2). Our overall results, show no apparent correlation in fadL and fadD mRNA expression levels and oleic acid uptake in E. coli. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.

Quantitative real-time PCR analysis

CityU_HK_project_result_fad1.png CityU_HK_project_result_fad2.png

Figure 1. Quantitative RT-PCR analysis of fadL and fadD mRNAs upon induction with IPTG a various concentrations. (A). Expression of fadL in control E. coli BL21(DE3) host cells (Blue) and fadL-fadD transformant (Red). (B). Expression of fadD in control E. coli BL21(DE3) host cells (Blue) and fadL-fadD transformant (Red). E. coli cells were cultured in LB + oleic acid (3.5 µM) medium and induced with 0 µM, 40 µM or 400 µM IPTG for 6 hrs. Cells were harvested for total RNA extraction and lipid extraction for GC-MS analyses.

GC-MS analysis

CityU_HK_project_result_fad3.png

Figure 2. Oleic acid concentration in fadL-fadD transformant and control E. coli cells at various concentrations of IPTG. Intracellular concentration of oleic acid in control E. coli BL21(DE3) host cells (Yellow) and fadL-fadD transformant (Gray). E. coli cells were incubated in LB + oleic acid (10 µM) medium and induced with 0 µM, 40 µM or 400 µM IPTG for 6 hrs.

Gas chromatography:

CityU_HK_fad_Gas_chromatography1.jpg CityU_HK_fad_Gas_chromatography2.jpg CityU_HK_fad_Gas_chromatography3.jpg CityU_HK_fad_Gas_chromatography4.jpg

Figure 3. Gas chromatography mass spectrum of fadL-fadD transformant and control cells: We expressed fadL-fadD cloned in the vector pSNAP-tag® (T7)-2 in E. coli BL21 (DE3) and the intracellular fatty acid concentrations of the recombinant cells was measured by GC-MS. (A) Chromatogram of control cells without IPTG. (B) Chromatogram of control cells with 400 μM IPTG. (C) Chromatogram of recombinant cells without IPTG (D) Chromatogram of recombinant cells with 400 μM IPTG. No significant difference in oleic acid concentration between control cells and recombinant cells without IPTG, but the recombinant cells showed two-fold higher oleic acid concentration than control cells.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1185
  • 1000
    COMPATIBLE WITH RFC[1000]


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