Prevention
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.
Detection
pET28a(+)-CP5, pET28a(+)-PPDK plasmid construction.
Recombinant CP5 production optimization.
Prevention
Preparation of E. coli Nissle and L. casei BL23 competent cells.
Genome editing strategy development.
Detection
Recombinant CP5 purification.
pET28a(+)-N-His, pET28a(+)-N-His-TEV plasmid construction.
Prevention
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
sgRNAs, recombination templates, primers construction in silico.
Detection
Recombinant CP5 refolding and activation.
Prevention
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
pTarget receiving from our advisor and multiplication in E. coli DH5 alpha strain.
Detection
Recombinant CP5 refolding and activation.
Prevention
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
Detection
ePCR preparation.
Recombinant CP5 refolding and activation.
Prevention
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
pLCNICK plasmid construction with sfGFP.
Detection
ePCR preparation.
Recombinant CP5 refolding and activation.
Recombinant PPDK production optimization.
Prevention
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.
Detection
Recombinant PPDK production optimization.
Prevention
pTRKH2+sfGFP and pTRKH2+loop+sfGFP construction for fluorescence measurements.
pTarget plasmid construction.
pLCNICK plasmid construction with sfGFP.
Detection
Recombinant PPDK solubilisation and purification.
Prevention
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.
Detection
Experimental PDA synthesis.
Recombinant and denatured PPDK and CP5 stabilization experiments.
Prevention
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.
Detection
Anti-HSA aptamer conjugation with PDA.
Testing of PDA with Anti-HSA aptamer.
Recombinant PPDK purification.
Prevention
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.
Detection
Protein purification with magnetic beads.
SELEX preparation, first round.
Prevention
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.
Detection
SELEX rounds 1-8.
Prevention
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.
Detection
Aptamer ligation into pUC19 for Sanger sequencing.
NGS preparation.
Prevention
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.
Detection
NGS preparation.
Prevention
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.
Detection
Sanger sequencing data analysis.
NGS preparation.
Prevention
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.
Detection
Sanger sequencing data analysis. NGS of aptamers.
Prevention
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.
Detection
NGS data analysis.
Prevention
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.
Detection
Anti-PPDK aptamer conjugation with PDA.
pTRKH2+TAL+4CL+CHS+CHI and pTRKH2+loop+TAL+4CL+CHS+CHI construction by Gibson assembly.
Prevention
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.
Detection
Diagnostic test assembly.
IT aptamer evaluation.