Difference between revisions of "Part:BBa K1469002"
 
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UDP-glucose 6-dehydrogenase of ''Bacillus megaterium''. The enzyme catalyses the NAD dependent oxidation of UDP-glucose to UDP-glucuronic acid, a key precursor molecule for hyaluronic acid production.  
 
UDP-glucose 6-dehydrogenase of ''Bacillus megaterium''. The enzyme catalyses the NAD dependent oxidation of UDP-glucose to UDP-glucuronic acid, a key precursor molecule for hyaluronic acid production.  
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=====Characterization by SSTi-SZGD(2018)=====
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<p>We characterized this part by co-overexpressing it with gtaB (<a href="https://parts.igem.org/Part:BBa_K1469005">BBa_K1469005</a>) together in an operon. UDP-GlcDH is also known as tuaD in B.subtilis, and its 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. Gene products of tuaD and gtaB regulate the last two steps in the synthetic pathway of UDP-GlcUA, one of the two precursors of HA. </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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===Usage and Biology===
 
===Usage and Biology===
  
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<partinfo>BBa_K1469002 parameters</partinfo>
 
<partinfo>BBa_K1469002 parameters</partinfo>
 
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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. <a href="https://parts.igem.org/Part:BBa_K2593008">The results showed</a> 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.
 

Latest revision as of 01:59, 18 October 2018

UDP-GlcDH

UDP-glucose 6-dehydrogenase of Bacillus megaterium. The enzyme catalyses the NAD dependent oxidation of UDP-glucose to UDP-glucuronic acid, a key precursor molecule for hyaluronic acid production.


Characterization by SSTi-SZGD(2018)

We characterized this part by co-overexpressing it with gtaB (BBa_K1469005) together in an operon. UDP-GlcDH is also known as tuaD in B.subtilis, and its 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. Gene products of tuaD and gtaB regulate the last two steps in the synthetic pathway of UDP-GlcUA, one of the two precursors of HA.


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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]