DNA

Part:BBa_K4737000

Designed by: Peisong Wu   Group: iGEM23_BNUZH-China   (2023-08-26)


prpsM

prpsM is the promoter of 30S ribosomal subunit protein S13 of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2


Usage and Biology

We used prpsM to start the expression of Lpp-OmpA-scFv, SopE-FLAG-GOx, TetR, T7 RNA polymerase, HlyA and so on.



design-figure-1.png
Figure 1: The Lpp-OmpA-scFv expression pathway.

design-figure-2new.png
Figure 2: The SopE-FLAG-GOx expression pathway.

design-figure-4.png
Figure 3: The HlyA expression pathway.

design-figure-6.png
Figure 4: The T7 RNA Polymerase expression pathway.



Recombinant S. typhimurium (VNP-SopE-GOx) was grown at appropriate conditions. The proteins from the culture supernatant and bacterial lysates were prepared for an immunoblotting assay. The results of Western blot showed that VNP20009 electroporated with plasmid pFPV25.1-SopE-FLAG-GOx expressing SopE-FLAG-GOx protein successfully (Figure 5).


gox-wb-bac.png
Figure 5: Western blot analysis shows the expression of gene of interest(SopE-FLAG-GOx) in VNP-SopE-GOx. The theoretical molecular weight of SopE-FLAG-GOx is about 76 kDa, and the theoretical molecular weight of GAPDH is about 36 kDa.




To verify the engineered bacteria can achieve the expression of HlyA, bacterial lysates were prepared from S. typhimurium strain VNP20009 carrying appropriate expression vectors for shRNA-SLC7A11 and subjected to immunoblotting analysis.HlyA expression detected by Western blotting indicates that our plasmids were successfully transferred into VNP20009 and effectively expressed (Figure 6).


shrna-result-3.png
Figure 6: Western blot analysis of Listeriolysin O. a.Listeriolysin O is a pore-forming protein (59kDa) encoded by gene HlyA b.GAPGH is a reference protein in cells with a molecular weight of 36 kDa.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1
    Illegal PstI site found at 34
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1
    Illegal PstI site found at 34
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1
    Illegal PstI site found at 34
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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