NextSeq 500 sequencing; Parallel monitoring of the integration efficiency and stability of mini-TLCΦ plasmids with different attachment sites
Parallel monitoring of the integration efficiency and stability of mini-TLCΦ plasmids with different attachment sites
FX498_s
Integration assays: V. cholerae recipients (CMV30 and EPV369) were grown to an OD600 of 0.3 in LB with 0.2% of L-arabinose (L-ara), to induce the Xer machinery. E. coli β2163 donors were grown to an OD600 of 0.6 in LB supplemented with 0.3 mM of Dap, and mixed with recipients at a 1:10 ratio. Donor and recipient cells were incubated for 3h on LB agar plates supplemented with Dap and L-ara, resuspended in LB supplemented with 0.2% of L-ara and incubated for an additional 1h. Conjugants were selected for the plasmid antibiotic resistance (chloramphenicol)and Dap autotrophy. In the case of the degenerate plasmid libraries, several conjugations were performed for each integration assay to ensure the recovery of ~10+6 clones.  Stability assays: Integration libraries were incubated without any selection pressure for the maintenance of the integrated elements. Fresh LB was inoculated with ~1x10+9 cells from the integration libraries, with the addition of arabinose 0.2%.  genomic DNA extraction kit Plasmid attP sites were amplified by performing 17 PCR cycles on 50-100 ng of plasmid library gDNA with an equimolar mix of 4103-4105 P5 and 4178-4180/4246-4248 P7 primers. The products were purified from the P5 and P7 primers the double selection with AMPure.