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.