Coding

Part:BBa_K1472601

Designed by: Lo Hiu Fung, Lau Tsz Kit, Chu Lok Ting, Wu Chun Ting   Group: iGEM14_CityU_HK   (2014-09-16)

Leaderless thioesterase

Escherichia coli thioesterase without a leader peptide sequence (‘TesA)

We have cloned a modified thioesterase gene, ‘tesA, which codes for an E. coli thioesterase protein that lacks the N-terminal 26-amino acid signal peptide sequence, and is named here as “leaderless TesA” or ‘TesA. The ‘TesA protein is expected to be expressed exclusively in the cytosol of E. coli to catalyze the conversion of fatty acyl-ACP (acyl carrier protein) or fatty acyl-CoA to free fatty acids (Figure 1). ‘TesA is predicted to enhance free fatty acid production in the genetically-modified E. coli strain.

CityU_HK_newBB_TesA_figure1.jpg

Figure 1. Conversion of fatty acyl-CoA or fatty acyl-ACP to free fatty acid.

Characterization :

We aimed to overexpress the ‘tesA gene (which codes for thioesterase I) in E. coli to (1) enhance the conversion of fatty acyl-CoA to free fatty acid, and (2) bypass the β-oxidation pathway for fatty acid metabolism. To facilitate this strategy, we have subcloned the ‘tesA coding sequence downstream of the PBAD promoter in the pSB1C3 vector. E. coli TOP10 cells (Invitrogen) was used as the host to overexpress ‘tesA. Expression of the ‘tesA gene was measured by quantitative RT-PCR and the effect of ‘TesA overexpression (leading to a possible increase in thioesterase I activity) was indirectly tested by determining the oleic acid concentrations in control and ‘tesA recombinant E. coli cells by GC-MS.

Results :

Quantitative real-time PCR analyses showed that ‘tesA mRNA overexpression was observed only in recombinant E. coli Top10 cells and not in the DH5α host. Expression of ‘tesA was 2.5-fold higher in the recombinant ‘tesA E. coli TOP10 cells than the control cells (Figure 1).


CityU_HK_project_result_tesA1.png"

Figure 1. Quantitative RT-PCR of ‘tesA expression in E. coli DH5α and TOP10 host cells. Expression of the ‘tesA gene was determined in control and ‘tesA recombinant E.coli DH5α and E. coli TOP10 cells. E. coli cells were cultured overnight in LB + oleic acid (3.5 mM) medium. Cells were harvested for total RNA extraction and lipid extraction for GC-MS analysis.

For the fatty acid analysis in E. coli TOP10 cells, the levels of both oleic acid and palmitic acid were elevated in recombinant ‘tesA TOP10 cells (but not recombinant DH5α cells) as compared to the control host cells (Figures 2A and 2B). The results suggested that the increase in oleic acid and palmitic acid levels in the recombinant TOP10 cells may be due to increased expression of the ‘tesA gene (and hence thioesterase I activity) in these E. coli cells. 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.




CityU_HK_project_result_tesA2.png"
CityU_HK_project_result_tesA3.png"

Figure 2. Intracellular levels of oleic acid and palmitic acid in recombinant ‘tesA and control E. coli cells. (A). Oleic acid concentration in control DH5α / TOP10 cells (Yellow), and ‘tesA recombinant DH5α /TOP10 cells (Gray). (B). Palmitic acid concentration in the control DH5α / TOP10 cells (Yellow), and ‘tesA recombinant DH5α /TOP10 cells (Gray).



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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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