Plasmid

Part:BBa_K5322032

Designed by: Xinquan Zhou   Group: iGEM24_NJTech-China   (2024-10-01)
Revision as of 13:54, 1 October 2024 by Baiyun (Talk | contribs)


Lpp-OmpA-Programmed Cell Death 1 Ligand 1 Functional Domain [Mus musculus] Expression System

Usage and Biology

During the project, we considered that PD-L1 might be encapsulated by mussel foot protein (Mfp) during expression and release, potentially reducing therapeutic efficacy. To address this, we utilized the surface display system Lpp-OmpA(BBa_K5322035) to present our passenger protein PD-L1 on the outer membrane of E. coli. This would facilitate its binding with PD-1, enhancing effectiveness. Additionally, to demonstrate surface display, we inserted a flexible protein linker and a TEV protease recognition site(BBa_K5322036) between Lpp-OmpA and the PD-L1 functional domain. By incubating the cells with TEV protease, PD-L1 could be cleaved from OmpA. Detection of the PD-L1 functional domain in the supernatant would indirectly confirm the success of surface display. We designed the plasmid pET29a-J23119-Lpp-OmpA-GISS-TEVsite-PD-L1(Functional domain)-T7.

pET29a-J23119-Lpp-OmpA-GISS-TEVsite-PD-L1(Functional domain)-T7

Figure 1-1 Plasmid pET29a-J23119-Lpp-OmpA-GISS-TEVsite-PD-L1(Functional domain)-T7

Construction of the plasmid

The target fragment was amplified by PCR, and the pET29a vector was amplified. The PCR product was subjected to gel electrophoresis (30 min, 120 V). After the product was purified, homologous recombination was performed and transformed into Escherichia coli DH5α. The plate was inverted and cultured at 37°C. After 16 hours, colony PCR was performed on a single colony. The results are shown in the figure.

Colony PCR

Figure 2-1 Colony PCR

The colony PCR was successfully inoculated at 37°C, 220rpm, 12-16h, the plasmid was extracted and sent to the company for sequencing. The sequencing results are shown in the figure below.

Sequencing results

Figure 2-2 Sequencing results

Protein characterization

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 832
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 790
    Illegal NheI site found at 1415
    Illegal PstI site found at 832
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 681
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 832
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 832
    Illegal NgoMIV site found at 1221
    Illegal AgeI site found at 1053
  • 1000
    COMPATIBLE WITH RFC[1000]
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