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 pACYCDuet-1-KpsE-KpsT was constructed, and the constructed plasmid and its empty vector were introduced into Nissle 1917 (DE3), respectively.
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 KpsE-KpsT genetically engineered bacterium showed a significant increase in pod production compared with the empty vector-imported bacterium.
Study:
As mentioned above, KpsE and KpsT 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
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2034
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 723 Illegal XhoI site found at 1968
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1196 Illegal SapI.rc site found at 1611
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