Difference between revisions of "Part:BBa K4325015"
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=2022 SZPT-China=
 
=2022 SZPT-China=
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<h3>Biology</h3>
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<h3>1.Characterization in <i>E. coli</i>  TOP10</h3>
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<p>This composite part is a generator consisting of a J23102 (<partinfo>BBa_J23102</partinfo>) promoter and gshF (<partinfo>BBa_K4325003</partinfo>).</p>
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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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<h3>Usage</h3>
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<p>The J23102 promoter and gshF were connected and inserted into the pSEVA331 expression vector so that gshF expressed the novel bifunctional enzyme GshF, which directly catalyze the synthesis of glutathione by the three amino acids of Cys, Glu and Gly.</p>
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[[File:K15 1a.png|600px|thumb|center|Figure 1: <p></p>(a) Verification of gshF in <i>E. coli</i> . <P></P>]]
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<h3>Characterization</h3>
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[[File:K15 1b.png|600px|thumb|center|Figure 1: <p></p>(b) Verification of SDS-PAGE electrophoresis in <i>E. coli</i> . <P></P>]]
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<h4><p>1.Verification of gshF in <i>E. coli</i> </p></h4>
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[[File:K15 1c.png|600px|thumb|center|Figure 1: <p></p>(c) Comparison of GSH production between wild type and engineered bacteria of <i> E. coli.</i> <P></P>]]
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<br>
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<h3>2.Characterization in <i>G. hansenii</i>  ATCC53582</h3>
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<p>Figure 2 (a) showed that the size of the DNA fragments amplified from G. hansenii, thus confirming the successful incorporation of the plasmid and Figure 2(b)showed that the GSH production in <i>G. hansenii</i> . <p>
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[[File:K3740044 Figure 2.png|600px|thumb|center|Figure 2: <p></p>(a) Growth curve of <i>E. coli</i>  TOP10 containing pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 #1<p></p>
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(b) Growth curve of <i>E. coli</i> TOP10 containing pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 #11]]

Revision as of 03:27, 10 October 2022


J23102-RBS003422-gshF-T0

Description

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


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 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.

Figure 1:

(a) Verification of gshF in E. coli .

Figure 1:

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

Figure 1:

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




2.Characterization in G. hansenii ATCC53582

Figure 2 (a) showed that the size of the DNA fragments amplified from G. hansenii, thus confirming the successful incorporation of the plasmid and Figure 2(b)showed that the GSH production in G. hansenii . <p>

Figure 2: <p>

(a) Growth curve of E. coli TOP10 containing pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 #1

(b) Growth curve of E. coli TOP10 containing pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 #11