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To enable co-expression with other genes, we selected pACYCDuet-1 and pETDuet-1 as our plasmid vectors. Through extracellular polysaccharide extraction experiments, both genes showed significant differences, successfully validating that our genes can promote the production of extracellular polysaccharides.
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Latest revision as of 15:30, 1 December 2024


pETDuet-1-galU-pgmA

To enable co-expression with other genes, we selected pACYCDuet-1 and pETDuet-1 as our plasmid vectors. Through extracellular polysaccharide extraction experiments, both genes showed significant differences, successfully validating that our genes can promote the production of extracellular polysaccharides.

  < img src="blob:chrome-untrusted://image-magnify/c657b01c-d703-4dda-8178-a0d2926475f6" style="display: block; margin: auto;width: 100%; ">  


Design: In podocarp research, the 2023 XJTU-iGEM team has used pgmA and galU gene overexpression to enable an increase in extracellular polysaccharide (EPS) production, based on which we additionally introduced overexpression of KpsE, KpsT, and FliC genes, which are capable of increasing the rate of podocarp polysaccharide translocation from intracellular to extracellular, thus optimizing our podocarp level of Defense.

Construct: The expression vector pETDuet-1-galU-pgmA was constructed, and the successfully constructed plasmid and its empty vector were introduced into Nissle 1917 (DE3) respectively.

  < img src="petduet-1-galu-pgma-plasmid-mapping.png" style="display: block; margin: auto;width:100%;height=auto;">

 

          Lane M: DNA Marker Lane 1: Plasmid digested by EcoRV and XhoI Lane 2: Uncut plasmid DNA    

 

Test: We carried out bacterial pod polysaccharide extraction on the above modified bacteria and measured the content of the extracted polysaccharide, using the empty vector-imported bacteria as the control, as shown in Fig. 1, our constructed PgmA-GalU genetically engineered bacterium showed a significant increase in pod production compared with the empty vector-imported bacterium.

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  < img src="pacycduet-1-kpse-kpst.png" style="display: block; margin: auto;width: 100%;height=auto; ">

 

          pETDuet-1-galU-pgmA when cultured to OD600=0.6, expression was induced by the addition of 0.5 mM IPTG for 3 h, and the bacterial broth was diluted to 0.4 at OD600    

 

Study: As mentioned above, PgmA-GalU genes can effectively increase pod yield, in the future, we will introduce pgmA and galU into EcN together with KpsE and KpsT genes, meanwhile, considering the possible influence of expression vectors on the expression of exogenous genes in chassis bacteria, we propose the idea of integrating exogenous genes genetically into EcN genome in order to eliminate the effect of expression vectors on the strain.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 166
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 166
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 166
    Illegal BamHI site found at 559
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 166
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 166
  • 1000
    COMPATIBLE WITH RFC[1000]