Part:BBa_K4841020

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

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

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: The plasmid map of pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SSRz-rrnB-Gox1

This mainly consists of two parts:

• 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.
• pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB backbone is the protein expression vector in this system. The red fluorescent protein mentioned above is expressed here. The CI857/PR (T41C) promoter is the heat shock promoter, and the maximum heat shock temperature can reach 380 degrees. The SRRz lysis cassette is extracted from phage and used for autolysis. pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB-Gox1 is the autolysis vector with added GOX1.

Figure 2: Scientific concept of heat-induced autolysis system

Experimental Approach and Construction Result

Figure 3: Electrophoresis gel results of pCOLA-CI857-SRRz-Gox1 plasmid construction.

After amplifying Gox1, we used the Gibson assembly to complete the construction of the pCOLA-CI857-SRRz-GOX1 vector. Colony PCR identification was performed to validate the successful construction.

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 BL21 E. coli, lysis was measured by the microplate reader and monitored to ensure enough lysis.

Figure 5: SDS-PAGE of pCOLA-CI857-SRRz-Gox1 plasmid protein expression after heat-induced autolysis.

After purifying samples from the lysis of pCOLA-CI857-SRRz-Gox1 BL21 E. coli, we performed SDS-PAGE to verify the expression of the target protein. The size of the target protein, Gox1, is 65kDa.

Characterization/Measurement

Protein expression, cell lysis, and SDS-PAGE

Figure 6: Glucose oxidase activity and CCK-8 activity of the GOX1 protein purified after heat-induced autolysis.

For the GOX1 protein purified after heat-induced autolysis, we also conducted glucose oxidase activity assay and CCK-8 test. Figure 4A shows that when the protein concentration increases, the glucose concentration increases in a positive trend, confirming that the protein is able to promote ferroptosis by consuming glutathione. The CCK-8 test in Figure 4B also backs up this inference.

Figure 7: GSH Activity test on 4T1 cells of the GOX1 protein purified after heat-induced autolysis.

GSH Activity Assay was also conducted with the GOX1 protein purified after heat-induced autolysis. Compared with the control group, as the protein concentration increases, the GSH content decreases. In other words, higher protein concentration possesses higher glutathione digestion performance, which also means a better effect on promoting 4T1 cell ferroptosis at the given range.

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