Difference between revisions of "Part:BBa K1469009"

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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.  
 
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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<img src="https://static.igem.org/mediawiki/parts/3/38/T--SSTi-SZGD--construct.jpeg"><br>
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<img src="https://static.igem.org/mediawiki/parts/c/cb/T--SSTi-SZGD--acid_pathway.png"style="width:50%">
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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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<img src="https://static.igem.org/mediawiki/parts/7/7d/T--SSTi-SZGD--p43nmk_acid.png"style="width:50%">
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<p>Fig1 a. the synthesis pathway of HA . b.the construct of glmU in pP43NMK plasmid </p><br>
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<p>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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<img src="https://static.igem.org/mediawiki/parts/6/6d/T--SSTi-SZGD--glmu_HA.png"style="width:50%">
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<img src="https://static.igem.org/mediawiki/parts/f/fa/T--SSTi-SZGD--coding_sequence.png"style="width:50%">
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<p>Figure 2: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of gtaB gene in pP43NMK-gtaB-tuaD using primer pair GlmU-F and GlmU-R and the expected product size is 304 bp. Right: illustration of construction of the expression vector pP43NMK-glmU. GlmU coding sequence was inserted at restriction sites HindIII and BamHI of pP43NMK plasmid.</p><br>
 
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<img src="https://static.igem.org/mediawiki/parts/3/38/T--SSTi-SZGD--construct.jpeg"><br>
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<img src="https://static.igem.org/mediawiki/parts/4/40/T--SSTi-SZGD--Illustration.png"style="width:50%">
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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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<img src="https://static.igem.org/mediawiki/parts/e/e4/T--SSTi-SZGD--CTAB_solution.jpeg"style="width:50%"></p>
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<p>Figure 3: CTAB analysis of HA concentration, 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:50%">
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<p>Figure4: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis. </p>
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<img src="https://static.igem.org/mediawiki/parts/c/c5/T--SSTi-SZGD--glmu_product.png"><br>
 
<img src="https://static.igem.org/mediawiki/parts/c/c5/T--SSTi-SZGD--glmu_product.png"><br>

Revision as of 17:07, 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.

Fig1 a. the synthesis pathway of HA . b.the construct of glmU in pP43NMK plasmid


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).

Figure 2: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of gtaB gene in pP43NMK-gtaB-tuaD using primer pair GlmU-F and GlmU-R and the expected product size is 304 bp. Right: illustration of construction of the expression vector pP43NMK-glmU. GlmU coding sequence was inserted at restriction sites HindIII and BamHI of pP43NMK plasmid.


Figure 3: CTAB analysis of HA concentration, 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.


Figure4: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.