Difference between revisions of "Part:BBa K1469009"

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Improvement Team:2018 SSTi-SZGD
 
 
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Revision as of 14:34, 17 October 2018


gcaD

UDP-N-acetylglucosamine pyrophosphorylase of Bacillus megaterium. The enzyme is bifunctional and catalyses the conversion of glucosamine 1-phosphate to N-acetyl glucosamine 1-phosphate as well as the conversion of N-acetyl glucosamine 1-phosphate to UDP-N-acetyl glucosamine, a key precursor molecule for hyaluronic acid production.

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-SZG(2018)

Usage

This is a improvement part of glmU (2014 saarland iGEM team) (also known as : GcaD in B.megaterium)

This device is used to over-express glmU gene in B. subtilis 168. glmU encodes UDP-N-acetylglucosamine pyrophosphorylase that converts GlcN-1-P to GlcNAc-1-P as well as GlcNAc-1-P to UDP-GlcNAc in the synthetic pathway of UDP-GlcNAc, one of the two HA precursors in B. subtilis. It is assumed that overexpression of glmU contributes to the elevation of UDP-GlcNAc production, which may have a positive effect on HA production.


In our project, glmU expression was regulated under the control of a constitutive promoter P43, and the recombinant pP43NMK-glmU vector was transformed into recombinant B.subtilis 168E, confirmed by colony PCR polymerization(Fig2) . It has been engineered to secrete extracellular HA. Our results showed the introduction of extra copies of glmU gene, resulted to an elevated HA accumulation in B.subtilis 168E (448mg/L, a 32% increase) (Fig3). Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Fig4).


In our project, glmU expression was regulated under the control of a constitutive promoter P43, and the recombinant pP43NMK-glmU vector was transformed into recombinant B.subtilis 168E, confirmed by colony PCR polymerization(Fig2) . It has been engineered to secrete extracellular HA. Our results showed the introduction of extra copies of glmU gene, resulted to an elevated HA accumulation in B.subtilis 168E (448mg/L, a 32% increase) (Fig3). Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Fig4).