Difference between revisions of "Part:BBa K4325015"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4325015 short</partinfo>
 
<partinfo>BBa_K4325015 short</partinfo>
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===Usage===
 
===Usage===
 
<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>
 
<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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[[File:K15 3.png|600px|thumb|center|Figure 1: <p></p> Gene circuit of J23102-RBS003422-gshF-T0. ]]
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===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-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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 +
 
 +
 
  
 
[[File:K15 1.png|600px|thumb|center|Figure 2: <p></p>(a) Verification of gshF in <i>E. coli</i> <p></p>
 
[[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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<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) 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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[[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 2b new.png|600px|thumb|center|Figure 3: <p></p>(a) Verification of gshF in <i>G. hansenii</i> ATCC53582.<p></p>
 
(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> . ]]
 
<h3>3.References</h3>
 
<h3>3.References</h3>
 
<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 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>

Revision as of 08:46, 10 October 2022


J23102-RBS003422-gshF-T0

Description

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

Usage

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.

Figure 1:

Gene 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-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.



Figure 2:

(a) Verification of gshF in E. coli

(b) Verification of SDS-PAGE electrophoresis in E. coli.

(c) Comparison of GSH production between wild type and engineered bacteria of E. coli .


2.Characterization in G. hansenii ATCC53582

Figure3(a) showed that the size of the DNA fragments amplified from G. hansenii , thus confirming the successful incorporation of the plasmid and Figure2(b) showed that the GSH production in G. hansenii.

Figure 3:

(a) Verification of gshF in G. hansenii ATCC53582.

(b) Comparison of GSH production between wild type and engineered G. hansenii .

3.References

[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.