Using Lambda Red Recombinase System for Gene Knockout
PFU PCR Linear DNA
The LacI and ArgI with 280 bp homology arm gene were amplify using PFU PCR.
LacI and ArgI Transformation into E. Coli W3100
Amplified PCR LacI and ArgI products were electroporated into E. Coli W3110 harboring pREDKI plasmid. The transformed cells were plated into a gradient of different spectinomycin concertation; starting at 60 ug/ml, 70 ug/ml and 80 ug/ml and 100 ug/ml.
Genomic PCR of the W3110 Lambda Red Modified Cells
A genomic PCR was done to confirm the recombination.
- ArgI Genomic PCR did not work in terms of Lambda Red Recombination because the gel still shows the native gene size of 1549bp not the ArgI Homology arms with the spectinomycin template which would be closer to 1995bp, but the gel reflects the size of just above 1500bp.
- LacI Genomic PCR did not work in terms of Lambda Red Recombination because the gel still shows the native gene size of 1616bp not the LacI Homology arms with the spectinomycin template which would be closer to 1990bp, but the gel reflects the size of just above 1500bp.
If the recombination worked an expect band size near the 2000bp mark on the ladder. Since the band size are the native gene size it is safe to assume that the cells didn't accept the linear gBlock for the recombination event.
The positive control was pREDKI which has a size of 1050 bp using the Exo Forward and Exo reverse primer sequences.
- Linear PCR fragments with the antibiotic template was frozen at -20C which could have caused issues because PCR products have nicks in the backbone.
- The antibiotic concentration for selection of the genomic recombination was not clear and so gradient of antibiotic was used and this lead to some false positive colonies on these plates.
- Concentration of arabinose for the pREDKI plasmid was not at the appropriate concentration for the most efficient expression of the lambda red recombination proteins.
- Optimize the pREDKI recombination protocol for higher efficiency.
- Knock out nonessential genes to metabolically engineer E. coli cells.
- Potentially knock in new genes to build a better metabolic pathway using other more efficient enzymes for putrescine production.
- Using pSIM plasmid as the biofunctional helper plasmid for the recombination
Amplification of Truncated ArgI sequence using PFU PCR protocol
The ArgI gene was amplify by PFU PCR using M13 primers.
The LacI gene was amplify by PFU PCR using T7 primers.
Digestion/Ligation of LacI and ArgI into pSB1C3
The truncated LacI and ArgI PCR product were digested with EcoRI-PstI and ligated with T4 ligase into pSB1C3. The ligation product was plated on 3.7 ug/ml chloramphenicol plates. Colonies were confirmed by direct colony PCR.