Difference between revisions of "Part:BBa K4325015"
 
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<partinfo>BBa_K4325015 short</partinfo>
 
<partinfo>BBa_K4325015 short</partinfo>
 
===Description===
 
===Description===
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The composite part is a generator consisting of a J23102 promoter and gshF.
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The composite part is a generator consisting of promoter J23102(<partinfo>BBa_J23102</partinfo>) and CDS gshF(<partinfo>BBa_K4325003</partinfo>).
  
 
===Usage===
 
===Usage===
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<p>The J23102 promoter and gshF were connected and inserted into the pSEVA331 expression vector so that gshF expressed the neotype  bifunctional enzyme GshF, which directly catalyze the synthesis of glutathione by the three kinds of amino acids, Cys, Glu and Gly.</p>
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<p>The promoter J23102(<partinfo>BBa_J23102</partinfo>),RBS003422(<partinfo>BBa_K4325006</partinfo>),CDS gshF(<partinfo>BBa_K4325003</partinfo>), and T0 terminator(<partinfo>BBa_K3257021</partinfo>) were connected and inserted into the pSEVA331 expression vector so that gshF expresses the bifunctional glutathione synthetase GshF, which directly catalyzes the synthesis of glutathione by the three kinds of amino acids, Cys, Glu and Gly.</p>
 +
[[File:K15 1.png|600px|thumb|center|Figure 1: Genetic circuit of J23102-RBS003422-gshF-T0. ]]
 +
 
 
===Sequence and Features===
 
===Sequence and Features===
  
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=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>
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<p>As shown in Figure 1, composite part J23102-RBS0034-gshF-T0 and the expression of GshF were verified successfully by PCR amplification and SDS-PAGE respectively. As well as GSH production was detected.</p>
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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> 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.]]
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[[File:K15 1.png|600px|thumb|center|Figure 2: <p></p>(a) Verification of gshF in <i>E. coli</i> <p></p>
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(b) Verification of SDS-PAGE electrophoresis in <i>E. coli.</i><p></p>
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(c) Comparison of GSH production between wild type and engineered bacteria of <i>E. coli</i> . ]]
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<br>
 
<br>
 
<h3>2.Characterization in <i>G. hansenii</i>  ATCC53582</h3>
 
<h3>2.Characterization in <i>G. hansenii</i>  ATCC53582</h3>
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<p>Figure2(a) showed that the size of the DNA fragments amplified from <i>G. hansenii</i> , thus confirming the successful incorporation of the plasmid and Figure2(b) showed that the GSH production in <i>G. hansenii.</i></p>
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<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 composite part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.</i></p>
  
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[[File:K15 2b new.png|600px|thumb|center|Figure 2: <p></p>(a) Verification of gshF in <i>G. hansenii</i> ATCC53582.<p></p>
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[[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>.]]
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(b) Comparison of GSH production between wild type and engineered <i>G. hansenii</i> . ]]
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<h3>References</h3>
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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>[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>
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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>

Latest revision as of 14:00, 12 October 2022

J23102-RBS003422-gshF-T0

Description

The composite part is a generator consisting of promoter J23102(BBa_J23102) and CDS gshF(BBa_K4325003).

Usage

The promoter J23102(BBa_J23102),RBS003422(BBa_K4325006),CDS gshF(BBa_K4325003), and T0 terminator(BBa_K3257021) were connected and inserted into the pSEVA331 expression vector so that gshF expresses the bifunctional glutathione synthetase GshF, which directly catalyzes 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 TOP10; (b) Verification of western blot electrophoresis in E. coli Top10; (c) Comparison of GSH production between wild type and engineered of 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 composite part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.</i>

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

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).