Plasmid

Part:BBa_K4841019

Designed by: Zhang Huixin   Group: iGEM23_TSBC-SZ   (2023-08-28)


pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB-Gox1

pACYC-pJ23117-esaR(I70V)-rrnBT-LSPT-esaI-BCD22-P8

Experiment Report

Engineering Principle and Design

To enable possible in vivo lysis progression and increase lysis efficiency and protein yield, a heat-induced autolysis pathway was designed and monitored with a quorum sensing system.

Figure 1
Figure 1: The plasmid map of pACYC-pJ23117-esaR(I70V)-rrnBT-LSPT-esaI-BCD22-P8
  • pACYC-pJ23117-esaR(I70V)-rrnBT-LSPT-esaI-BCD22-P8 plasmid is the sensor plasmid of the quorum sensing system. The esaI in the plasmid expresses the AHL factor, and the esaR (I70V) promoter is responsible for controlling the expression of the AHL factor. The expression of the AHL factor can induce the PesaS promoter to express a red fluorescent protein.
Figure 2
Figure 2: Scientific concept of heat-induced autolysis system

Experimental Approach and Construction Result

Figure 1
Figure 3: pACYC-pJ23117-esaR(I70V)-rrnBT-LSPT-esaI-BCD22-P8 plasmid construction.

After amplifying Gox1 in pCOLA vector, we used the Gibson assembly to complete the construction of the pACYC-pJ23117-esaR(I70V)-rrnBT-LSPT-esaI-BCD22-P8 vector. Colony PCR identification was performed to validate the successful construction.

Figure 2
Figure 4: Lysis result of heat-induced autolysis plasmid (pCOLA-CI857-SRRz-GOX1 and PaCYC-QS).

After overnight incubation of the pCOLA-CI857-SRRz-Gox1+pACYC-QS BL21 E. coli, lysis was measured by the microplate reader and monitored to ensure enough lysis.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4111
    Illegal NheI site found at 5340
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 206
    Illegal BamHI site found at 954
    Illegal XhoI site found at 2154
    Illegal XhoI site found at 4006
    Illegal XhoI site found at 4117
    Illegal XhoI site found at 5989
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4325
    Illegal BsaI site found at 6147
    Illegal BsaI site found at 6478
    Illegal BsaI.rc site found at 6424
    Illegal BsaI.rc site found at 7249


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