2.3. LASSO probe assembly
Pre-LASSO cloning in pLASSO . The fusion of the pre-LASSO probe in
pLASSO vector was performed by using NEBuilder HiFI DNA assembly (NEB).
By adjusting with PCR grade water to 20 µl total volume the following
components: Linearized pLASSO (~50 ng), Pre-LASSO probe
pool ~16 ng, Linearized pLASSO ~50 ng,
NEBuilderHiFi DNA Assembly Master Mix 2X 10μL. The solution was
Incubated in a PCR thermal cycler at 50°C for 15 minutes.
The NEBulder reaction 1µl was added to 50 μl of electrocompetent 5-alpha
E. coli cells (NEB) into a chilled 1 mm cuvette (BioRad) and
electroporated using a Micro Pulser (BioRad). The transformed cells were
recovered in 950μl optimal broth medium and were all plated on a 150 mm
petri dish containing 100 μg ml−1 ampicillin. The
numbers of single colonies were estimated by serial dilutions.
Depending on the number of colonies obtained, the electroporation was
repeated n times in order to obtain approximately 10 times coverage of
the E.coli library (~30k colonies)
All colonies from the petri dishes were recovered, resuspended in 5 ml
LB medium and subjected to plasmid extraction (Miniprep Kit, Qiagen).
The DNA concentration was measured with a Nanodrop.
The successful cloning of pre-LASSO library in pLASSO was verified by
digesting in 50µl total volume of 1X CutSmart Buffer, 500ng of the
recovered pLASSO library with 20 units of SalI and 20 units BamHI.
Digestion was performed for 1h at 37°C. The digestion solution (4µl) was
loaded on a 2% agarose EtBr gel and subjected to electrophoresis and
observed with a trans UV. If DNA band correspondent with the size of the
pre-LASSO library (~160bp) was present in the lane, the
pre-LASSO library was considered successfully cloned in pLASSO.
Cre-recombination. The pLASSO library (2µg) was subjected to
nicking endonuclease digestion in a PCR tube in 50µl of 1X Cut Smart
Buffer with 20 units of Nt.BbvCI nicking endonuclease (NEB). The
digestion was performed in a PCR thermal cycler for 1h at 37°C and heat
inactivated for 10 min at 80°C. The nicked unpurified pLASSO library
(250ng, correspondent to 6.25µl) were directly added into a PCR tube
containing 37.75 µl of nuclease free water, 5µl of CRE recombinase
buffer (NEB) and 1µl of Cre-recombinase (ABCAM) added last. The
recombination reaction was performed at 37°C for 30 min and heat
inactivate at 70⁰C for 10min. The temperature was then lowered to 25°C
and 1µl (20 units) of SwaI resctiction enzyme (NEB) were added directly
into the recombination solution. The digestion was carried on for 1h at
25°C and heat-inactivated at 70°C for 10 min. At this point, the
temperature was lowered again to 37 °C and 2µl ATP 10mM and 1µl (10
units) of Exonuclease V (NEB) were added into solution, mixed by
pipetting up and down, incubated at 37⁰C for 30min and heat-inactivate
at 70⁰C for 30min.
Inverted PCR . Inverted PCR was performed in a 50 μl total volume
containing 10 μl of the unpurified Cre-recombination-digestion reaction
as described above, 10 μl of 5x KAPA HiFi Fidelity Buffer, 0.3 mM each
dNTP, and 0.3 μM reverse primer ThioIR and 0.3 μM forward primer Sap1F
and 1 μl of KAPA HiFi HotStart DNA Polymerase. Both SapI and ThiolR
anneal with opposite orientations in the inverted PCR primer annealing
site central section of the pre-LASSO probe
(AACACTTCTTGCGGCGATGGTTCCTGGCTCTTCGATC). The first three bases of ThioIR
were phosphorothioate bonds to prevent digestion during subsequent ExoI
treatment, and a 3′ -terminal uracil base for subsequent primer removal
using Uracil-DNA Glycosylase (USER enzyme); Sap1F includes the SapI
restriction site (Type IIS side-cutting restriction enzyme) for primer
removal via digest with the isoschizomer BspQI. The PCR thermal profile
was initiated for 3 min at 95 °C, followed by 18 cycles of 20 s at 98
°C, 15 s at 60 °C and 60 s at 72 °C, and then 4 min at 72 °C.
The size of the inverted PCR product was purified using AMPure beads
(0.8×), washed with 80% ethanol twice and eluted with 25 μl of
nuclease-free water. The concentration of purified inverted PCR product
was measured by Nanodrop.
Production of mature LASSO probes . Approximately 200 ng of
purified inverted PCR product was digested by adding in a total volume
of 25 μl, 2.5 μl of 10× CutSmart buffer (NEB) and 1 μl of BspQI
restriction enzyme (NEB). Digestion was performed at 50 °C for 1 h
followed by 20 min at 80 °C. After digestion, 1 μl (five units) of
Lambda Exonuclease was added directly to the BspQI digested DNA and
incubated for 30 min at 37 °C followed by 10 min at 80 °C. At this
point, 2 μl (1 unit μl-1) of USER enzyme (NEB) was
added in solution and incubated for 30 min at 37 °C.