Part:BBa_K4737012
pnirB-HlyA-6xHis
To express HlyA-6×His in the anaerobic environment
Usage and Biology
While anti-CEA scFv can enhance the targeting ability of VNP20009 towards tumor cells, we aim to mitigate the toxic effects of this bacterium on normal tissue. To further enhance the safety of our project, we need to screen specific promoters that activate exclusively within the tumor microenvironment (TME). In solid tumors, the tumor tissue grows rapidly, leading to significant inflammation, and the vascular system within the tissue is incomplete. Consequently, the tumor microenvironment often exhibits characteristics indicative of overall oxygen deficiency. The nirB promoter is the promoter of the first gene of E. coli NADH-dependent nitrite reductase operon. This promoter is induced in anaerobic conditions and up regulated in response to nitrite and nitrate. Its synthesis mechanism has been thoroughly studied and has been chosen by numerous iGEM teams (e.g., Nanjing_China_Bio 2012, TecMonterrey 2013, etc.) to mitigate the toxic effects of bacteria on normal tissue. Based on the aforementioned considerations, we have selected the nirB promoter to initiate subsequent killing modules.
Figure 1. The anaerobic expression of Listeriolysin-O.
1. Plasmids construction
To enhance verification of the functioning of nirB promoter in VNP20009, we constructed the pnirB::HlyA-6×His plasmid (Fig.2)
Fig.2.The pnirB::HlyA-6×His vector that was constructed for verifying the function of pnirB.
Fig.3. Agarose gel electrophoresis analysis of linearized vector pFPV25.1.
Fig.4. Agarose gel electrophoresis analysis of pnirB (407 bp) and HlyA-6×His (1608 bp). Red frames outlined in the figure are target fragments. a. pnirB fragment; b. HlyA-6×His fragment.
We obtained the linearized vector by double digestion of vector pFPV25.1 with endonucleases XbaI and MfeI (Fig.3). We amplified the target fragment pnirB from E.coli K12 genome (Fig.4a) and the HlyA-6×His fragment synthesized by the company by PCR (Fig.4b), after which we connected above target fragments to the linearized vector through homologous recombination to obtain positive clone recombinants. Then we carried out colony PCR (Fig.5) and sanger sequencing to verify whether the HlyA-6×His was ligated into the vector, which meant pnirB::HlyA-6×His plasmid was constructed successfully.
Fig.5. Colony PCR were used to confirmed whether pnirB::HlyA-6×His was successfully ligated with the vector.
We transform the constructed plasmids into E.coli for incubating, then use alkaline lysis method to get new plasmids and transform them into VNP20009 respectively with electroporation.
2. Expression of effector proteins in S. typhimurium driven by pnirB.
Western blotting proved that engineered bacteria can be induced to produce Listeriolysin O under hypoxic conditions(Fig.6). We added a 6×His tag to the C-terminus of Listeriolysin-O on the vector, and the expression of Listeriolysin-O was characterized through it.
Fig.6. SDS-PAGE analysis of the expression of Listeriolysin-O. 6×His tag molecular weight is about 59.5 kDa and GAPDH is a reference protein in cells with a molecular weight of 36 kDa.
Based on above results, the engineered bacteria we constructed was successfully expressed target protein under hypoxic conditions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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