Difference between revisions of "Part:BBa K1469009"
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=====Improvement=====
 
=====Improvement=====
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2018 SSTi-SZGD erified that glmU increases HA production. glmU encodes a UDP-N-acetylglucosamine pyrophosphorylase.  it has 495 amino aicd   (also know as : GcaD in megaterium)<br>
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2018 SSTi-SZGD:
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168E-glmU produces precipitates formed by the reaction of HA with CTAB, OD400:2.313. HA’s concentration is 447.2mg/L.
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<html>
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<body>
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<h4>Usage</h4>
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<P>
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This is a improvement part of glmU (2014  saarland  iGEM team) (also known as : GcaD in <i>B.megaterium</i>)
 +
</P>
 +
<p>
 +
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.
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</p>
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<p>
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<img src="https://static.igem.org/mediawiki/parts/3/38/T--SSTi-SZGD--construct.jpeg"><br>
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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).
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</p>
 +
<p>
 +
<img src="https://static.igem.org/mediawiki/parts/3/38/T--SSTi-SZGD--construct.jpeg"><br>
 +
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).
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</p>
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<p>
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<img src="https://static.igem.org/mediawiki/parts/c/c5/T--SSTi-SZGD--glmu_product.png"><br>
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<img src="https://static.igem.org/mediawiki/parts/b/ba/T--SSTi-SZGD--analysis_of_HA.png"><br>
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<img src="https://static.igem.org/mediawiki/parts/c/cc/T--SSTi-SZGD--viscometer.png">
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</p>
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</body>
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</html>

Revision as of 10:09, 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


Improvement

2018 SSTi-SZGD:


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).