Difference between revisions of "Part:BBa K4325003"
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__NOTOC__
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<partinfo>BBa_K4325015 short</partinfo>
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===Description===
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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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===Sequence and Features===
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<partinfo>BBa_K4325015 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4325015 parameters</partinfo>
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<!-- -->
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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>
 +
<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>
 +
<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>
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 +
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4325003 short</partinfo>
 
<partinfo>BBa_K4325003 short</partinfo>
 
===Description===
 
===Description===
−
<i> gshF </i>encodes a bifunctional glutathione synthetase GshF originated from <i> Streptococcus thermophilus</i>  
+
<i> gshF encodes a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus.</i>  
 
===Usage===
 
===Usage===
−
<p>it is less sensitive to GSH, is selected and codon optimized for expressing in <i>G. hansenii</i>.  
+
<p>It is less sensitive to GSH, is selected and codon optimized for expressing in <i>G. hansenii</i></i>..
  
 
===Sequence and Features===
 
===Sequence and Features===
Line 22: Line 65:
 
As shown in Figure 1, the expression of GshF were verified successfully by Western blot.</p>
 
As shown in Figure 1, the expression of GshF were verified successfully by Western blot.</p>
 
<h4>2.References</h4>
 
<h4>2.References</h4>
−
<p>[1]Li W1,Li Z,Yang J,Ye Q, et al.Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli.Journal of Biotechnology, 12 Jun 2011, 154(4):261-268.</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 <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>

Revision as of 11:08, 12 October 2022

J23102-RBS003422-gshF-T0

Description

The composite part is a generator consisting of promoter J23102 and CDS gshF.

Usage

The promoter J23102BBa_J23102 and CDS gshFBBa_K4325003 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.

Figure 1: Genetic circuit of J23102-RBS003422-gshF-T0.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 223
    Illegal EcoRI site found at 805
    Illegal EcoRI site found at 1240
    Illegal EcoRI site found at 1738
    Illegal PstI site found at 64
    Illegal PstI site found at 94
    Illegal PstI site found at 451
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 223
    Illegal EcoRI site found at 805
    Illegal EcoRI site found at 1240
    Illegal EcoRI site found at 1738
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal PstI site found at 64
    Illegal PstI site found at 94
    Illegal PstI site found at 451
    Illegal NotI site found at 1883
    Illegal NotI site found at 2083
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 223
    Illegal EcoRI site found at 805
    Illegal EcoRI site found at 1240
    Illegal EcoRI site found at 1738
    Illegal XhoI site found at 2064
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 223
    Illegal EcoRI site found at 805
    Illegal EcoRI site found at 1240
    Illegal EcoRI site found at 1738
    Illegal PstI site found at 64
    Illegal PstI site found at 94
    Illegal PstI site found at 451
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 223
    Illegal EcoRI site found at 805
    Illegal EcoRI site found at 1240
    Illegal EcoRI site found at 1738
    Illegal PstI site found at 64
    Illegal PstI site found at 94
    Illegal PstI site found at 451
    Illegal NgoMIV site found at 1665
    Illegal NgoMIV site found at 2296
  • 1000
    COMPATIBLE WITH RFC[1000]


2022 SZPT-China

1.Characterization in E. coli TOP10

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 E. coli TOP10 containing this composite part is much higher (~92 fold) than that of wild-type E. coli TOP10..

Figure 2: (a) Verification of gshF in E. coli; (b) Verification of western blot electrophoresis in E. coli Top10; (c) Comparison of GSH production between wild type and engineered bacteria of E. coli Top10.


2.Characterization in G. hansenii ATCC53582

Figure3 (a) showed the DNA fragments amplified from G. hansenii, thus confirming the successful incorporation of the plasmid. Figure2 (b) showed that G. hansenii containing this composite part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.</i>

Figure 3: Verification of gshF in G. hansenii ATCC53582; (b) Comparison of GSH production between wild type and engineered G. hansenii; (b) Comparison of GSH production between wild type and engineered G. hansenii.

3.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).






gshF

Description

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).