Difference between revisions of "Part:BBa K1469005"
 
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<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
===Functional Parameters===
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=====Improvement=====
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2018 SSTi-SZGD coexpress tuaD and gtaB together, because gtab and tuad are two of the genes involved in the synthesis of UDP-glucose dehydrogenase and UDP-gulcose pyrophosphorylase. The results showed that co-expressing of tuaD and gtaB genes increased HA accumulation, suggesting that UDP-glucose dehydrogenase and UDP-gulcose pyrophosphorylase, which commit to the biosynthesis of one of the HA precursors, are rate-limiting enzymes in HA biosynthesis.
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=====Characterization by SSTi-SZGD(2018)=====
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<p>This is an improvement of this part and tuaD(UDP-GlcDH )(<a href="https://parts.igem.org/Part:BBa_K1469002">BBa_K1469002</a>), which were originally introduced by saarland iGEM in 2014. We characterized this part by co-overexpressing it with tuaD(<a href="https://parts.igem.org/Part:BBa_K1469002">BBa_K1469002</a>)together in an operon, it’s gene product may contribute positively to HA production. </p><br>
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<p>In our project, expression of the operon tuaD-gtaB was regulated under the control of a constitutive promoter P43, this operon is used for further increasing the production of the HA in B.subtilis, tuaD and gtaB gene, products regulate the last two steps in the synthetic pathway of UDP-GlcUA.</p><br>
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<img src="https://static.igem.org/mediawiki/parts/c/cb/T--SSTi-SZGD--acid_pathway.png"style="width:40%">
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<img src="https://static.igem.org/mediawiki/parts/7/7d/T--SSTi-SZGD--p43nmk_acid.png" style="width:40%">
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<p>Fig1  the synthesis pathway of HA .
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In our experiment, by conducting CTAB experiments that form turbidity from a reaction between HA and CTAB solution, the results showed a remarkable increase in HA production when co-overexpressed tuaD-gtaB together (488mg/L, a 38% increase) (Figure2),In addition, Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Figure3 ). </p>
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<img src="https://static.igem.org/mediawiki/parts/e/e4/T--SSTi-SZGD--CTAB_solution.jpeg"style="width:40%">
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<p>Figure 2: CTAB analysis of HA concentraton, a. Illustration of the turbidity by mixing different source of HA with CTAB solution. b: effects of overexpressing the precursor genes on HA production in recombinant B. </p><br>
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<img src="https://static.igem.org/mediawiki/parts/4/4c/T--SSTi-SZGD--viscometer_analysis.png" style="width:40%">
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<p>Figure 3: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.  </p>
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Latest revision as of 02:42, 18 October 2018

gtaB

UTP--glucose-1-phosphate uridylyltransferase of Bacillus megaterium. The enzyme catalyzes the conversion of glucose 1-phospahate to UDP-glucose 1-phosphate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 768
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 220



Characterization by SSTi-SZGD(2018)

This is an improvement of this part and tuaD(UDP-GlcDH )(BBa_K1469002), which were originally introduced by saarland iGEM in 2014. We characterized this part by co-overexpressing it with tuaD(BBa_K1469002)together in an operon, it’s gene product may contribute positively to HA production.


In our project, expression of the operon tuaD-gtaB was regulated under the control of a constitutive promoter P43, this operon is used for further increasing the production of the HA in B.subtilis, tuaD and gtaB gene, products regulate the last two steps in the synthetic pathway of UDP-GlcUA.


Fig1 the synthesis pathway of HA . In our experiment, by conducting CTAB experiments that form turbidity from a reaction between HA and CTAB solution, the results showed a remarkable increase in HA production when co-overexpressed tuaD-gtaB together (488mg/L, a 38% increase) (Figure2),In addition, Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Figure3 ).

Figure 2: CTAB analysis of HA concentraton, a. Illustration of the turbidity by mixing different source of HA with CTAB solution. b: effects of overexpressing the precursor genes on HA production in recombinant B.


Figure 3: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.