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	__NOTOC__		__NOTOC__
−	<partinfo>BBa_K4325015 short</partinfo>	+	<partinfo>BBa_K4325003 short</partinfo>
	===Description===		===Description===
−	The composite part is a generator consisting of promoter J23102 and CDS gshF.	+	<p><i>gshF</i> encodes a bifunctional glutathione synthetase GshF originated from <i>Streptococcus thermophilus</i>.</p>
−		+
	===Usage===		===Usage===
−	<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>	+	<p>It is less sensitive to GSH, is and codon optimized for expressing in <i>G. hansenii</i>.
−	[[File:K15 1.png|600px|thumb|center|Figure 1: Genetic circuit of J23102-RBS003422-gshF-T0. ]]	+
−		+
	===Sequence and Features===		===Sequence and Features===
−	<partinfo>BBa_K4325015 SequenceAndFeatures</partinfo>	+	<partinfo>BBa_K4325003 SequenceAndFeatures</partinfo>
	<!-- Uncomment this to enable Functional Parameter display		<!-- Uncomment this to enable Functional Parameter display
	===Functional Parameters===		===Functional Parameters===
−	<partinfo>BBa_K4325015 parameters</partinfo>	+	<partinfo>BBa_K4325025 parameters</partinfo>
	<!-- -->		<!-- -->
	=2022 SZPT-China=		=2022 SZPT-China=
	<h3>1.Characterization in <i>E. coli</i>  TOP10</h3>		<h3>1.Characterization in <i>E. coli</i>  TOP10</h3>
−	<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>	+	As shown in Figure 2, the expression of GshF which encoded by BBa_K4325003 were verified successfully by PCR amplification and western blot respectively. The GSH production of <i>E. coli</i> TOP10 containing this part is much higher (~92 fold) than that of wild-type <i>E. coli</i> TOP10.</p>
−	[[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.]]	+	[[File:K15 2.png|800px|thumb|center|Figure 2: (a) Verification of gshF in <i>E. coli</i> TOP10; (b) Verification of western blot electrophoresis in <i>E. coli</i> Top10; (c) Comparison of GSH production between wild type and engineered of <i>E. coli</i> Top10.]]
	<br>		<br>
	<h3>2.Characterization in <i>G. hansenii</i>  ATCC53582</h3>		<h3>2.Characterization in <i>G. hansenii</i>  ATCC53582</h3>
−	<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>	+	<p>Figure3 (a) shows the DNA fragments of gshF were amplified from <i>G. hansenii</i>, thus confirming the successful incorporation of the plasmid. Figure2 (b) shows that <i>G. hansenii</i> containing this part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.</p>
−	[[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>.]]	+	[[File:K15 3.png|600px|thumb|center|Figure 3: (a) Verification of gshF in <i>G. hansenii</i> ATCC53582; (b) Comparison of GSH production between wild type and engineered <i>G. hansenii</i>.]]
−	<h3>3.References</h3>	+	<h3>References</h3>
	<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>		<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>
	<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>		<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>[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>		<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>
	<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>		<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>
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−	__NOTOC__
−	<partinfo>BBa_K4325003 short</partinfo>
−	===Description===
−	<i> gshF encodes a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus.</i>
−	===Usage===
−	<p>It is less sensitive to GSH, is selected and codon optimized for expressing in <i>G. hansenii</i></i>..
−
−	===Sequence and Features===
−
−	<partinfo>BBa_K4325003 SequenceAndFeatures</partinfo>
−
−
−	<!-- Uncomment this to enable Functional Parameter display
−	===Functional Parameters===
−	<partinfo>BBa_K4325025 parameters</partinfo>
−	<!-- -->
−	=2022 SZPT-China=
−	<h3>Characterization</h3>
−	<h4>
−	1. Verification of Western blot electrophoresis in <i> E. coli</i>.</h4>
−	[[File:Mfold-K3257013-1.png|600px|thumb|center|Figure1: Verification of Western blot electrophoresis in <i> E. coli.</i>]]
−	As shown in Figure 1, the expression of GshF were verified successfully by Western blot.</p>
−	<h4>2.References</h4>
−	<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 <i>Escherichia coli</i>. J. Biotechnol. <b>154</b>, 261-268 (2011).</p>
−	<p>[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).</p>

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Latest revision as of 17:15, 13 October 2022

gshF

Description

gshF encodes a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus.

Usage

It is less sensitive to GSH, is and codon optimized for expressing in G. hansenii.

Sequence and Features

2022 SZPT-China

1.Characterization in E. coli TOP10

As shown in Figure 2, the expression of GshF which encoded by BBa_K4325003 were verified successfully by PCR amplification and western blot respectively. The GSH production of E. coli TOP10 containing this part is much higher (~92 fold) than that of wild-type E. coli TOP10.

2.Characterization in G. hansenii ATCC53582

Figure3 (a) shows the DNA fragments of gshF were amplified from G. hansenii, thus confirming the successful incorporation of the plasmid. Figure2 (b) shows that G. hansenii containing this part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.

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] Wang, D., Wang, C., Wu, H., Li, Z. & Ye, Q. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase. J. Ind. Microbiol. Biotechnol. 43, 45-53 (2016).

[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. 123, 194-203 (2017).

[4] Xiong, Z.-Q. et al. Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. J. Dairy Sci.101 , 6937-6945 (2018).

[5] Cui, X. et al. Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters. J. Biotechnol.289, 39-45 (2019).