Composite

Part:BBa_K4644018

Designed by: Han Cao   Group: iGEM23_BUCT   (2023-10-10)


pCS-lac-grosl

        In E. coli expression systems, inclusion bodies can form when expressing foreign proteins due to the lack of modification systems. Currently, the mainstream approach in the scientific community to increase solubility is to purify and refold the expressed proteins or use molecular chaperones or solubilization tags. Therefore, BUCT has attempted four molecular chaperones to enhance solubility: GroEL/GroES, DnaK/DnaJ, IbpAB, and sigma32. This plasmid containing GroEL/GroES sequence.

Fig 4. pcs-lac-grosl


Solubility Verification

        The gene segments constructed as described above were subjected to homologous recombination with the pET-duet plasmid using the same method. E. coli BL21 cells, which had been pre-cultured and stored, were revived. The plasmids were transformed into the host cells through electroporation. Four different molecular chaperones were separately introduced into the host cells, and a control group with an empty vector and another control group with no molecular chaperones were included, resulting in a total of six groups.After selection for antibiotic resistance, the cells were initially inoculated into LB medium containing both ampicillin and kanamycin antibiotics. Subsequently, they were transferred to 50 ml of LB medium containing both antibiotics in shake flasks and cultured at 37°C until the OD reached the desired range (0.2-0.6). The cultures were induced with 0.5 mM IPTG and further incubated for 12-16 hours.The fermentation broth was then collected and subjected to repeated sonication for cell disruption. Each group was divided into two fractions: the supernatant and the precipitate. Finally, the gel electrophoresis results for this protein were obtained using SDS-PAGE.


Fig 8. Urease SDS-PAGE-(1)

Fig 9. Urease SDS-PAGE-(2)

After comparison, it is easy to notice that the effect of GroEL/GroES

Reference

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 88
    Illegal PstI site found at 203
    Illegal PstI site found at 702
    Illegal PstI site found at 828
    Illegal PstI site found at 998
    Illegal PstI site found at 1316
    Illegal PstI site found at 1544
    Illegal PstI site found at 1961
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 88
    Illegal PstI site found at 203
    Illegal PstI site found at 702
    Illegal PstI site found at 828
    Illegal PstI site found at 998
    Illegal PstI site found at 1316
    Illegal PstI site found at 1544
    Illegal PstI site found at 1961
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 88
    Illegal BamHI site found at 2051
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 88
    Illegal PstI site found at 203
    Illegal PstI site found at 702
    Illegal PstI site found at 828
    Illegal PstI site found at 998
    Illegal PstI site found at 1316
    Illegal PstI site found at 1544
    Illegal PstI site found at 1961
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 88
    Illegal PstI site found at 203
    Illegal PstI site found at 702
    Illegal PstI site found at 828
    Illegal PstI site found at 998
    Illegal PstI site found at 1316
    Illegal PstI site found at 1544
    Illegal PstI site found at 1961
    Illegal AgeI site found at 992
  • 1000
    COMPATIBLE WITH RFC[1000]


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