Amplification culture and strain preservation of Cellulomonas fimi (CF).

Extract the whole genome DNA of CF.

Amplify the Cex and CenA genes of CF by PCR and amplify the signal peptide by PCR.

Detect Cex and CenA by agarose gel electrophoresis, extract PCR products Cex and CenA, and fusion of signal peptides and target fragments by Overlap PCR.

Extract AAV plasmids pHelper and pAAV6.

Amplify LL-37 and bFGF gene fragments synthesized by the company, identify PCR products by agarose gel electrophoresis, purify of PCR products.

Expansion of Gluconacetobacter xylinum.

Amplify the Cex and CenA genes of CF by PCR and amplify the signal peptide by PCR.

Detect Cex and CenA by agarose gel electrophoresis, extract PCR products Cex and CenA, and fusion of signal peptides and target fragments by Overlap PCR.

Extract AAV plasmids pHelper and pAAV6.

Amplify LL-37 and bFGF gene fragments synthesized by the company, identify PCR products by agarose gel electrophoresis, purify PCR products.

Double enzyme digestion of pAAV6 plasmid, agarose gel electrophoresis to detect the size of gene fragments, and cut agarose gel to extract gene fragments.

Multi batch of BC film production, sterilization, and pretreatment before coculture.

Extract red light control plasmid REDMAP.

Extract AAV plasmids pHelper and pAAV6.

Amplify LL-37 and bFGF gene fragments synthesized by the company, identify PCR products by agarose gel electrophoresis, purify PCR products.

Double enzyme digestion of pAAV6 plasmid, agarose gel electrophoresis to detect the size of gene fragments, and cut agarose gel to extract gene fragments.

Construct recombinant plasmid by ligating LL-37 and pAAV6 plasmid with T4 DNA ligase and construct recombinant plasmid by joining bFGF with pAAV6 plasmid by homologous recombination.

Resuscitate fibroblast ATCC CRL-2522 (BJ), BGC823 cells, and HEK293T cells.

Coculture of three kinds of cells (BGC823, HEK293T, BJ) with BC membrane.

Extract lentivirus packaging plasmids psPAX2 and pMD2.G.

Amplify Bglx gene fragments synthesized by the company, agarose gel electrophoresis to detect the size of gene fragments, and cut agarose gel to purify PCR products, fusion of signal peptides and target fragments by Overlap PCR.

Extract the blue light empty plasmid (PLVX) synthesized by the company.

Extract AAV plasmids pHelper and pAAV6.

Amplify LL-37 and bFGF gene fragments synthesized by the company, identify PCR products by agarose gel electrophoresis, purify PCR products.

Double enzyme digestion of pAAV6 plasmid, agarose gel electrophoresis to detect the size of gene fragments, and cut agarose gel to extract gene fragments.

Construct recombinant plasmid by ligating LL-37 and pAAV6 plasmid with T4 DNA ligase and construct recombinant plasmid by joining bFGF with pAAV6 plasmid by homologous recombination.

Extract lentivirus packaging plasmids psPAX2 and pMD2.G.

Transforming competent cells with psPAX2 and pMD2.G plasmids.

Double enzyme digestion of PLVX and assembling three target gene (Cex、CenA、Bglx) fragments with PLVX plasmid using homologous recombination to obtain three recombinant plasmids (PLVX-Cex, PLVX-CenA, PLVX-Bglx) of the Blue light system.

Sequencing and confirmation of the recombinant plasmids.

Amplify bFGF-6xHis gene fragment and extract plasmids (pAAV6-LL-37, pAAV6-EGF, and pAAV6-bFGF).

Transiently transfect recombinant plasmid into HEK293T and BGC823.

Proteinswere detected by SDS-PAGE electrophoresis, coomassie brilliant blue staining/ Western blot.

Extract blue light controlled recombinant plasmids PLVX-Cex, PLVX-CenA, and PLVX-Bglx.

Transfect the recombinant plasmids into HEK293T.

Proteinswere detected by SDS-PAGE electrophoresis, coomassie brilliant blue staining/ Western blot.

Test blue light induced BC degradation.

pAAV6-LL-37/pAAV6-EGF/pAAV6-bFGF and pHelper plasmids were transfected into HEK293T cells to pack AAV.

Collect cells, split cells to obtain viruses, purify viruses, and verify packaging by qPCR.

ELISA validation of the expression of EGF/LL-37 in the cell culture supernatant.

Verify that red light induces cell suicide.

The MTT assay was used to verify the red light suicide system.

REDMAP, psPAX2, pMD2.G plasmids were transfected into cells to pack lentivirus.

qPCR validation of whether lentivirus was successfully produced.

The recombinant plasmids were transfected into cells to pack lentivirus.

Collect virus supernatant.

Infection of HEK293T with virus supernatant.

Screening cells with puromycin.

Infect HEK293T and BGC823 with successfully packaged viruses.

Screening cells with G418.