mutant generation final steps

assays/Mutant Generation/protocols/R401andEcoliConjugation.md

+## Conjugation of E. coli and R401 and selection for first homologous recombination event
+
+*E. coli* BW29427 cells containing the plasmid and R401 were inoculated into 4 ml of 50% TSB containing 25 μg/ml Kan and 50 μg/ml DAP or 50% TSB and incubated overnight at 37 °C with 180 rpm agitation or 25 °C with 180 rpm agitation, respectively. Cells were harvested by centrifugation at 8,000 rpm for 2 min at room temperature, then washed 3x and subsequently resuspended in 1 ml of 50% TSB followed by centrifugation, after which the supernatant was discarded. After quantifying OD600, both cultures were mixed to equal parts and approx. 10x concentrated by centrifugation. The bacterial suspension was plated on 50% TSA plates containing 50 μg/ml DAP and incubated at 25 °C overnight to allow for conjugation events. The mating patches were scraped of the plate and resuspended in 1 ml 50% TSB. Then, 100 μl were spread on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Nitrofurantoin (Nitro; Sigma-Aldrich; to counter-select *E. coli*) and incubated at 25 °C. Colonies were validated for successful genomic insertion of the plasmid via colony PCR using a primer specific to the genomic DNA approx. 150 bp upstream of the upward flanking region (upup) and the plasmid specific M13F primer. Colony PCR was performed on at least 15 separate colonies and a WT control with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously, but with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis followed by Sanger sequencing following the manufacturer's protocol.
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assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md

+## Sucrose counter-selection to induce the second homologous recombination event
+
+A R401 colony with a successful genomic insertion of the plasmid was resuspended from a plate into 1 ml of 50% TSB. The cell density in the medium was then measured using the Multisizer 4e Coulter Counter (Beckman Coulter) following the manufacturer's protocol. One hundred microliters of 500 cells/μl, 5,000 cells/μl and 50,000 cells/μl dilutions were spread on three separate 50% TSA plates containing 300 mM sucrose. The plates were incubated at 25 °C for approx. 48 h. At least 30 colonies were examined by colony PCR using the respective upup and dwdw primers. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Positive colonies were purified by streaking on new 50% TSA plates and further verified by Sanger sequencing (Eurofins Scientific) following the manufacturer's protocol. They were also streaked on 50% TSA containing 25 μg/ml Kan to verify loss of the plasmid. A second colony PCR was performed on positive colonies and a wt control to validate the absence of the GOI, using a forward (inF) and reverse (inR) primer inside the GOI. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25 μl reactions as described previously. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Upon successful verification, 4 ml of 50% TSB were inoculated with a positive colony and grown overnight at 25 °C at 180 rpm. Finally, 750 μl of the overnight culture were added to 750 μl of 50% glycerol in an internally threaded 1.8 ml Nunc CryoTube, gently mixed, and stored at -80 °C.
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