The composite part is a generator consisting of promoter J23102 and CDS gshF.
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===Usage===
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<p>The promoter J23102<partinfo>BBa_J23102</partinfo> and CDS gshF<partinfo>BBa_K4325003</partinfo> were connected and inserted into the pSEVA331 expression vector so that gshF expressed the bifunctional glutathione synthetase GshF, which directly catalyze the synthesis of glutathione by the three kinds of amino acids, Cys, Glu and Gly.</p>
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[[File:K15 1.png|600px|thumb|center|Figure 1: Genetic circuit of J23102-RBS003422-gshF-T0. ]]
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===Functional Parameters===
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<partinfo>BBa_K4325015 parameters</partinfo>
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=2022 SZPT-China=
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<h3>1.Characterization in <i>E. coli</i> TOP10</h3>
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<p>As shown in Figure 2, composite part J23102-RBS003422-gshF-T0 and the expression of GshF were verified successfully by PCR amplification and western blot respectively. The GSH production of <i>E. coli</i> TOP10 containing this composite part is much higher (~92 fold) than that of wild-type <i>E. coli</i> TOP10..</p>
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[[File:K15 2.png|800px|thumb|center|Figure 2: (a) Verification of gshF in <i>E. coli</i>; (b) Verification of western blot electrophoresis in <i>E. coli</i> Top10; (c) Comparison of GSH production between wild type and engineered bacteria of <i>E. coli</i> Top10.]]
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<br>
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<h3>2.Characterization in <i>G. hansenii</i> ATCC53582</h3>
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<p>Figure3 (a) showed the DNA fragments amplified from <i>G. hansenii</i>, thus confirming the successful incorporation of the plasmid. Figure2 (b) showed that <i>G. hansenii</i> containing this composite part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.</i></p>
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[[File:K15 3.png|600px|thumb|center|Figure 3: Verification of gshF in <i>G. hansenii</i> ATCC53582; (b) Comparison of GSH production between wild type and engineered <i>G. hansenii</i>; (b) Comparison of GSH production between wild type and engineered <i>G. hansenii</i>.]]
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<h3>3.References</h3>
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<p>[1] Li, W., Li, Z., Yang, J. & Ye, Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. J. Biotechnol. <b>154</b>, 261-268 (2011.</p>
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<p>[2] Wang, D., Wang, C., Wu, H., Li, Z. & Ye, Q. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase. J. Ind. Microbiol. Biotechnol. <b>43</b>, 45-53 (2016).</p>
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<p>[3] Pophaly, S. D. et al. Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). J. Appl. Microbiol. <b>123</b>, 194-203 (2017).</p>
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<p>[4] Xiong, Z.-Q. et al. Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. J. Dairy Sci.<b>101</b> , 6937-6945 (2018).</p>
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<p>[5] Cui, X. et al. Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters. J. Biotechnol.<b>289</b>, 39-45 (2019).</p>
gshF encodes a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus.
Usage
It is less sensitive to GSH, is selected and codon optimized for expressing in G. hansenii</i>..
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 178 Illegal EcoRI site found at 760 Illegal EcoRI site found at 1195 Illegal EcoRI site found at 1693 Illegal PstI site found at 19 Illegal PstI site found at 49 Illegal PstI site found at 406
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 178 Illegal EcoRI site found at 760 Illegal EcoRI site found at 1195 Illegal EcoRI site found at 1693 Illegal PstI site found at 19 Illegal PstI site found at 49 Illegal PstI site found at 406 Illegal NotI site found at 1838 Illegal NotI site found at 2038
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 178 Illegal EcoRI site found at 760 Illegal EcoRI site found at 1195 Illegal EcoRI site found at 1693 Illegal XhoI site found at 2019
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 178 Illegal EcoRI site found at 760 Illegal EcoRI site found at 1195 Illegal EcoRI site found at 1693 Illegal PstI site found at 19 Illegal PstI site found at 49 Illegal PstI site found at 406
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 178 Illegal EcoRI site found at 760 Illegal EcoRI site found at 1195 Illegal EcoRI site found at 1693 Illegal PstI site found at 19 Illegal PstI site found at 49 Illegal PstI site found at 406 Illegal NgoMIV site found at 1620 Illegal NgoMIV site found at 2251
1000
COMPATIBLE WITH RFC[1000]
2022 SZPT-China
Characterization
1. Verification of Western blot electrophoresis in E. coli.
Figure1: Verification of Western blot electrophoresis in E. coli.
As shown in Figure 1, the expression of GshF were verified successfully by Western blot.
2.References
[1] Li, W., Li, Z., Yang, J. & Ye, Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. J. Biotechnol. 154, 261-268 (2011).
[2] Pophaly, S. D. et al. Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). J. Appl. Microbiol. 123, 194-203 (2017).
