2.1. pLASSO vector generation and linearization
pLASSO vector was derived from the linear pLox2+ (NEB). 50 ng of
pLox2+ were circularized by adding 1 µl of T4 DNA ligase (NEB), 1X T4
DNA ligase buffer, nuclease-free water to 25 µl total volume and
incubated overnight at 16 ⁰C. The ligation reaction (0.5µl) was used to
transform 5-alpha chemically competent E. coli cells (NEB).E.coli colonies were collected from ampicillin agar plates after
overnight incubation. Single colonies (four) were inoculate in 5 ml
corning tubes containing Ampicillin LB medium and shacked at 200 RPM ON
at 37 ⁰C.
The cells were pelleted and subjected to plasmid extraction by using the
PureLink Quick Plasmid Miniprep Kit (Invitrogen) as described by the
vendor and checked for the presence of pLox2+ by running 2µl of the
plasmid miniprep in a 0.8% agarose gel with Sybr Green. The pLox2+
plasmids (500ng) was digested with 20 units of EcoRI enzyme (NEB) and 5
units of Alkaline Phosphatase (NEB) in 25µl of 1X CutSmart buffer (NEB),
Incubate in the thermal cycler at 37⁰C for 1h and heat inactivate at
80⁰C for 10min. The digested pLox2+ (200 ng) was run on a Sybr Green
agarose gel (1%), visualized at the blue light and the DNA band
correspondent with the expected 2866 bp fragment was cut from the gel
with a scalpel and DNA extracted using
Monarch
DNA Gel Extraction Kit (NEB).
100ng of the synthetic dsDNA fragment Backbone (gBlocks, IDT) were
digested with 20 unit of EcoRI restriction enzyme in 25ul of 1X CutSmart
buffer at 37C for one hour and purified by using “DNA Purification SPRI
Magnetic Bead as described by the vendor. The EcoRI digested Backbone
(10ng) were mixed with 50ng of the the 2866 bp fragment obtained from
the EcoRI digestion of pLox 2+ in 25µl volume of 1X T4 DNA ligase buffer
and n units of T4 DNA ligase. Ligation was performed overnight at 16°C.
0.5 µL of the ON ligation were used for transformation of 5-alpha
chemically competent E. coli cells NEB and plated on an Ampicillin
resistance selective agar plate. Colonies were harvested from Ampicillin
agar plates and grown overnight in LB broth (5 ml). The pLASSO plasmid
was extracted with PureLink Quick Plasmid Miniprep Kit as described by
the vendor from pelleted broth cultures. The identity of pLASSO was
verified cutting with single or combination of the SalI, BamHI, SwaI and
EcoRI restriction enzymes and checking expected DNA band sizes on
agarose gel according with pLASSO map (Supplementary material
1 ).
Finally, pLASSO plasmid was linearized by performing an inverted PCR in
a 25µl of 1X Kapa Hi Fidelity Buffer, pLASSO ( 0.5ng), dNTPS (0.3 mM)
and 0.5 units of Kapa Taq DNA polymerase, and Linearization primers (0.3
µM). NEB1F and NEB1R. The thermal profile was: initial denaturation 4min
at 95°C, ( 95°C for 20 sec, 60°C for 20 sec, 2 min at 72°C) for 28
cycles, and final extension 3 min at 72°C