Recovery of Escherichia coli Nissle and Lactobacillus casei BL23 cultures.
Searching for the most suitable and effective E. coli Nissle 1917 and L. casei BL23 genome editing strategy.
pET28a(+)-CP5, pET28a(+)-PPDK plasmid construction.
Recombinant CP5 production optimization
Preparation of E. coli Nissle and L. casei BL23 competent cells.
Genome editing strategy development.
Recombinant CP5 purification.
pET28a(+)-N-His, pET28a(+)-N-His-TEV plasmid construction.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
sgRNAs, recombination templates, primers construction in silico.
Recombinant CP5 refolding and activation.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
pTarget receiving from our advisor and multiplication in E. coli DH5 alpha strain.
Recombinant CP5 refolding and activation.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
ePCR preparation.
Recombinant CP5 refolding and activation.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
pLCNICK plasmid construction with sfGFP.
ePCR preparation.
Recombinant CP5 refolding and activation.
Recombinant PPDK production optimization.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
sgRNA and gRNA oligos annealing and ligation with pTarget plasmids.
Construction of recombination template with colicin homology arms and sfGFP insertion, reactions’ conditions optimizations.
pLCNICK plasmid construction with sfGFP.
Recombinant PPDK production optimization.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
pTarget plasmid construction.
pLCNICK plasmid construction with sfGFP.
Recombinant PPDK solubilisation and purification.
pTRKH2+sfGFP and pTRKH2+loop+sfGFP fluorescence measurements.
Terminator insertion to the pTRKH2+loop system.
pTarget amplification, purification and preparation for sequencing.
Colicin, adhE, ackA, pta, tyrP PCR from genomic DNA optimization.
Experimental PDA synthesis.
Recombinant and denatured PPDK and CP5 stabilization experiments.
pTRKH2+TAL/4CL/CHS/CHI and pTRKH2+loop+TAL/4CL/CHS/CHI construction.
Analysis of ackA, pta, adhE, tyrP genes sequencing results and previously designed sgRNA validation.
Anti-HSA aptamer conjugation with PDA.
Testing of PDA with Anti-HSA aptamer.
Recombinant PPDK purification.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.
pCas9 plasmid amplification.
Nissle electroporation with pCas9.
sfGFP insertion into L. casei.
Protein purification with magnetic beads.
SELEX preparation, first round.
pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP construction.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.
sfGFP insertion into colicin gene in E. coli Nissle 1917.
sfGFP insertion into L. casei.
SELEX rounds 1-8.
VapXD system construction.
pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP construction.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.
AckA, pta, adhE, tyrP knockouts creation.
Aptamer ligation into pUC19 for Sanger sequencing.
NGS preparation.
VapXD system construction.
pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP construction.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.
AckA, pta, adhE, tyrP knockouts and their combinations creation.
NGS preparation.
VapXD serial dilution spotting.
pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP fluorescence measurements.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.
Fusion protein construction.
AckA, pta, adhE, tyrP knockouts and their combinations creation.
Sanger sequencing data analysis.
NGS preparation.
pTRKH2+TAL-sfGFP and pTRKH2+loop+TAL-sfGFP fluorescence measurements.
VapXD serial dilution spotting.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction.
AckA, pta, adhE, tyrP knockouts and their combinations creation.
VapXD system insertion into colicin gene in E. coli Nissle.
Sanger sequencing data analysis. NGS of aptamers.
Plate reader measurements of VapXD construct in pTRKH2 plasmid.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.
AckA, pta, adhE, tyrP knockouts and their combinations creation.
NGS data analysis.
Fusion protein construction.
New crRNA (for tyrP, adhE, nupG) annealing with gRNA and ligation into pTarget.
Recombination template construction for VapXD system insertion into nupG gene.
sfGFP insertion into wild type, new knockouts combinations creation with newly obtained sgRNA.
Genes of naringenin synthesis enzymes insertion into colicin and nupG genes into wild type and ackA-pta knockout strains.
Anti-PPDK aptamer conjugation with PDA.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.
Plate reader measurements of VapXD construct in E. coli Nissle 1917 genome.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.
Genes of naringenin synthesis enzymes insertion into colicin and nupG genes into wild type and ackA-pta knockout strains.
Intermediate compounds of naringenin synthesis from E. coli Nissle 1917 growth medium and cell lysate identification by MS.
Diagnostic test assembly.
IT aptamer evaluation.