3.2. Cre-recombinase mediated assembly of LASSO probe libraries
In order to assess the effectiveness of our novel DNA recombinase mediated assembly methodology in producing complex LASSO probe libraries, we performed a comparison to our previous work. In this previous project, we had designed pre-LASSO probes to target 3164 ORFs from E.coli genomic DNA (Tosi et al 2017). We maintained the previous central inverse PCR primer annealing site while we modified the previous pre-LASSO probe DNA ends for entry in pLASSO vector (Fig. 1 a,a and b termini). Analogously to what was reported by Tosi et al. (2017), out of the ~ 4000 E.coli K2 annotated ORFs we removed pre-LASSO probes corresponding to ORF targets smaller than 400 bp as a precaution to avoid potentially skewing our capture library during its subsequent PCR amplification and we also removed additional 160 probes that targeted different capture targets’ lengths as negative controls. Adjusting the thresholds for target length, melting temperature or the length of the ligation / extension arms determines the number of acceptable probes to 3078. As a result of these filters, approximately 22.5% of the E. coli K12 ORFeome (900 ORFs) was thus left untargeted and is used as an internal, negative control for our experiments. The ssDNA pre-LASSO library was obtained as a pre-LASSO probe oligo pool with each oligo at a size of ~ 160bp. The E.coli LASSO probe library was assembled as shown in Fig. 2 . The pre-LASSO ssDNA oligo pool was converted into dsDNA format by PCR (Fig. 2a ), and cloned in the linearized pLASSO (Fig. 2b ). The pLASSO library obtained was expanded by transformation in E.coli cells and harvested from antibiotic selective agar plates (Fig. 2b) . The presence of the pre-LASSO library in the pLASSO plasmids was verified by performing double digestion with SalI and BamHI restriction enzymes (Fig. 1b ). Gel electrophoresis (Fig. 2c ) showed a ~ 170bp band indicating the pre-LASSO library was successfully cloned in pLASSO.
The library was subsequently treated with Cre recombinase enzyme to create DNA mini-circles with LASSO probe precursors Interestingly, this recombination could not occur in the native supercoiled state of the plasmid library and required relaxation of the structure. Plasmids were converted in the relaxed form by digesting with the nicking endonuclease Nt.BvC1 that produces a DNA nick in correspondence of a restriction site located in the backbone (Fig. 2 d ). The products of Cre-mediated recombination at loxP sites are DNA minicircles containing the pre-LASSO probes and 2.7 kb DNA circles containing the remaining part of pLASSO (Fig. 2 e ). The agarose gel electrophoretic run of the recombination reaction (Fig. 2 f illustrates successful formation of the expected DNA minicircles (orange arrow) together with the 2.7 kb circular DNA remaining parts of pLASSO (green arrow). Since Cre-mediated recombinase enzyme requires no energy cofactors and quickly reaches equilibrium between substrate and reaction products, a DNA band corresponding to the size of the pLASSO substrate (blue arrow) was expectedly observed.