of 5
Cloning guide for
the
pTS93
-
116
lentiviral
transfer
plasmid
toolbox
The pTS93
-
116 lentiviral transfer plasmid toolbox
was created to allow easy implementation of
our anti
-
GFP/ALFA nanobody
-
based purification strategy. It
contains plasmids that allow fusion
of a protein of interest (POI) to either N
-
or C
-
terminal GFP or ALFA peptide tags with or without
additional protease cleavage sites. This guide describes how to clone your POI into these
plasmids
to create a
transfer p
lasmid
that
can be co
-
transfected with the packaging plasmids
psPAX2 (
Addgene #12260)
and pMD2.G (
Addgene #12259)
into Lenti
-
X
293T
cells
to create
lentivirus
.
The resulting lentiviral supernatant can then be used to transduce
for example
human
Expi
293F suspension cells to express your tagged POI for purification trials.
Figure 1.
(Top) Schematic of pTS93, with detailed sequence view of the
multi
ple
cloning site (MCS)
for
pTS93
-
pTS102. (Bottom) Schematic of p
T
S103, with detailed sequence view of the MCS for pTS103
-
116 (Kozak sequence underlined).
5
’ LTR =
5
l
ong terminal repeat, RRE = Rev response element, cPPT
= central polypurine tract, CMV
TetO2
= cytomegalovirus promoter with 2x TetO
elements
, MCS =
multipl
e cloning site, WPRE = Woodchuck hepatitis virus post
-
transcripti
o
nal regulatory element,
3
’ LTR
=
3
’ long terminal repeat,
Amp
R
= Beta
-
lactamase (ampicil
l
in/carben
i
ci
l
lin resistance),
ORI = origin of
replication
All
transfer
plasmids contain the same multiple cloning site (MCS)
with
4 unique restriction sites
(
Fig. 1
)
.
Using the same
restriction site
, multiple plasmids encoding the POI fused to either GFP
or ALFA tag at N
-
or C
-
terminus can therefore
easily
be generated
d
urin
g initial optimization trials.
Besides restriction enzyme digest, w
e routinely clone
POIs
into these plasmids using
G
ibson
assembly
.
To choose
a
method, consider the following:
Restriction enzyme cloning
Gibson cloning
Template
DNA
features
Insert
sequence needs to lack the
chosen
MCS
restriction sites
Internal restriction sites in
insert don’t interfere
Modularity
Modular, digested i
nsert
fragment
can
be
ligate
d
into any plasmid
Not modular, d
ifferent primers
needed to clone
insert
into
different
backbones
Speed
Restriction
digest
adds extra step
after PCR
There’s no
need to digest
the
PCR product
Primer
length
Requires shorter primer
overhangs
(6 bp flanking + 6
bp restriction
site)
Requires longer primer
overhangs (15
-
40 bp Gibson
overhangs)
General p
lasmid design considerations
:
Make
sure to include a stop codon in your reverse primer when cloning into pTS93
-
pTS102
.
T
here is a stop codon after the MCS but at
least 2 amino acids will be added if no stop codon
is added to the insert fragment
.
When adding an N
-
terminal tag (
using
pTS93
-
pTS102) check the
U
niprot annotation for your
POI to see if the initiator methionine is removed. If so, it is best to avoid including this codon
in your insert
.
When adding a C
-
terminal tag, the N
-
terminus of your
POI will include additional amino acids
from the MCS sequence as well as a
n initiator
methionine that we have included in the
backbone to ensure a functional
K
ozak
consensus
sequence
(5’
-
(gcc)gccRcc
ATG
G
-
3’)
.
If it is important to
maintain
the
endogenous N
-
terminus, you
can
digest your backbone with
NotI
,
but you must ensure that the resulting sequence contains a functional
K
ozak sequence.
Restriction enzyme cloning
Choosing r
estriction
sites:
1.
Check the sequence of your
POI
DNA
template
for
BamHI, NheI, AgeI, and XbaI
recognition
sites
and choose
2 sites in the MCS that
do
not
occur
in your
template
.
2.
If possible,
choose the outermost sites to
avoid adding excess amino acids to the N
-
or C
-
terminus of your POI (see
Fig.
1
)
.
3.
A
void using
both
NheI and XbaI when possible because these two enzymes
create
matching
overhangs
. This thus
reduc
es
the chan
c
e of successful ligation
in the correct orientation
.
Designing primers:
1.
Make sure
that you
are using the correct reading frame of the template DNA and that it does
not contain premature stop codons.
2.
Design both
forward and reverse primers
to
be between 20
-
40 b
ase
p
airs (bp)
in length and
to
have
a melting temperature (T
M
)
between
5
8
-
64
.
M
ake
sure each primer
ends in
a
G
or
C
at the
3’ end
.
3.
The 5’ end of each primer must contain a string of 6 nucleotides (of any sequence) followed
by the restriction site. This is needed to provide a toehold for the restriction enzyme.
4.
Design
the
reverse
prime
r
to be reverse complement of the template DNA sequence
.
5.
Check
primers for both intra
-
and intermolecular
high T
M
hairpins using
e.g.
IDT
’s
oligoanalyzer (
https://www.idtdna.com/pages/tools/oligoanalyzer
)
and introduce changes to
remove these if necessary
.
Run PCR reaction:
1.
Mix reaction components in a 0.2 ml PCR tube on ice
PCR reaction
(50
μl)
Volume (μl)
Reagent
25
Q5
High
-
Fidelity
2x
M
aster
M
ix (NEB,
USA)
1
10 ng/μl template
0
.5
5
0 μM forward primer
0
.5
5
0 μM reverse primer
23
ddH
2
O
2.
Program
thermocycler with the
following
settings and run PCR
:
Thermocycler settings
#
Step
Temperature
Time
1
Initial denaturation
98°C
30 sec
2
3
4
Denaturation
Annealing
Extension
98°C
58
-
64°C
72°C
10 sec
30 sec
30 sec per 1 kbp
30x
cycles of
steps 2
-
4
5
Hold
4
-
10°C
kbp = kilo base pair
3.
Purify reactions using Q
IAquick
PCR
Purification
k
it (QIAGEN, Netherlands)
4.
Elute in 42 μl ddH
2
O
Double restriction enzyme digest of purified PCR product:
1.
Set up restriction digest reactions of
purified
PCR product and plasmid:
PCR product digest reaction (50 μl)
Plasmid digest reaction (50 μl)
Volume
(μl)
Reagent
5
10x CutSmart buffer (NEB, USA)
1.5
Restriction enzyme 1
*
(30 Units)
1.5
Restriction enzyme 2
*
(30 Units)
42
P
urified PCR product
*
Use of NEB high fidelity (HF) enzymes
is
recommended
2.
Mix
well
and i
ncubate
digests
at 37
̊
C for
1.5
h
3.
Add 2 μl
of 1U/μl
FastAP
Alkaline phosphatase
(Thermo Fisher Scientific, USA)
only
to
the
plasmid digest
to remove 5’ phosphates (prevents self
-
ligation of insert
-
less plasmid)
4.
Incubate
both digests
for another 30 min at 37 ̊C
5.
Mix
digests
with 10 μl
Gel
L
oading
D
ye
, Purple (6x)
(NEB, USA)
6.
Run on a 1%
(w/v)
agarose gel
made up
in 1x TAE buffer
and supplemente
d with 1x
SYBR
S
afe DNA
Gel S
tain (Thermo Fisher
Scientific
, USA)
for 30 min at 150 V
7.
Excise band
s
with clean razor blade
s
8.
Purify excised DNA band
s
with Zymoclean Gel DNA
R
ecovery
K
it (Zymo Research, USA)
9.
Elute
in two steps
with 2x 10
μl ddH
2
O
(20 μl final volume)
10.
Measure DNA concentration
using a
N
ano
D
rop
spectrophotometer (Thermo Fisher Scientific,
USA)
Ligation
Volume
(μl)
Reagent
5
10x CutSmart
buffer (NEB, USA)
1.5
Restriction enzyme 1
*
(30 Units)
1.5
Restriction enzyme 2
*
(30 Units)
X
5 μg plasmid DNA
Add to 50
ddH
2
O
1.
Using the size
in bp
of both digested insert and
plasmid
fragments, calculate the amount of
insert in ng to ligate with 100 ng deph
osphorylated
plasmid using
a 2:1 insert:plasmid ratio
à
U
se th
e
NEBioCalculator
:
https://nebiocalculator.neb.com/#!/ligation
2.
Set up the ligation reactio
n and include a negative control reaction in which
the
insert is
replaced with
ddH
2
O
Ligation reaction (10 μl)
Volume (μl)
Reagent
5
2
x
Quick
L
igase
Reaction Buffer
X
X ng of insert
DNA
X
100 ng of
plasmid DNA
0.5
Quick
ligase
(NEB, USA
)
Add to
10
ddH
2
O
3.
Mix
well
and incubate for
15
min at room temperature
Transformation
1.
Thaw one 100 μl vial of chemical
ly
competent
E. coli
Stellar cells (Takara Bio, Japan)
per two
ligation reactions for 10 min on ice
2.
Split into 2x 50 μl aliquots in two separate
tubes on ice and add 5 μl of ligation reaction to
each
3.
Incubate for 30 min on ice
4.
Heat shock tubes at 42 ̊C for 35 sec in a heat block or water bath
5.
Quickly remove to ice and chill for 1
-
2 min
6.
Rescue by addition of
200
μl SOC recovery medium
7.
Incubate
tubes
at 37 ̊C for 30
-
60 min shaking at 1,200 rpm
8.
Plate out ̃150 μl
on LB
-
Carb
agar
plates, let dry and incubate
upside down
overnight at 37 ̊C
Gibson cloning
Primer design
Use NEBuilder
Assembly tool to design insert primers with matching overhangs for Gibson
assembly:
https://nebuilder.neb.com/#!/
1.
Generate
a
new fragment and copy&paste backbone plasmid DNA sequence, click process
text a
nd check the ‘vector’ and ‘circular’ boxes. Rename
‘new fragment’
to ‘plasmid’.
2.
Select ‘restriction digest’ as the method for production of a linearized fragment and specify
your choice of restriction sites. Finally click ‘Add’.
3.
Generate
another
new fragme
nt and copy&paste insert template DNA sequence, click
process text and check the ‘vector’ and ‘circular’ boxes if applicable. Rename to ‘insert’.
4.
Select ‘PCR’ as the method for production of a linearized fragment and specify the start and
end base of your
insert.
Include stop codon if needed.
Finally click ‘Add’.
5.
The exonuclease in the Gibson assembly mix will remove the 4 bp overhangs generated by
restriction digest of the plasmid, leaving only 1 base of the original 6 bp recognition site behind.
In order
to maintain a proper open reading frame
,
upstream and
/or
downstream junctions
between plasmid and insert fragments
may
need to be adjusted
by adding either two or five
bases, to restore a single codon or the complete restriction site, respectively
. Click o
n
the
pencil symbol of the newly added insert fragment to edit
these junctions
.
6.
The program generates an ‘assembled sequence’ that should be thoroughly inspected to
contain the insert at the correct location and in the correct reading frame.
7.
If all looks
well order the suggested insert primer pair containing the proper Gibson assembly
overhangs.
PCR
amplification of insert fragment
1.
Set up and run PCRs as described above
Restriction
digest
to create plasmid fragment
1.
While the insert PCR is running, set up
the plasmid restriction digest reaction as described
above and include the phosphatase treatment step
Gel purification
1.
Mix
insert PCR
and plasmid restriction digest reaction
with 10 μl
Gel
L
oading
D
ye
, Purple (6x)
(NEB, USA)
, r
un on a 1% (w/v) agarose an
d purify from excised gel band as described above
Gibson
assembly
reaction
1.
Calculate the volume of insert
and vector fragment that contains 50 fmoles of each using
NEBioCalculator:
https://nebiocalculator.neb.com/#!/dsdnaamt
2.
Mix calculated volume of both vector and insert fragment and then dilute two
-
fold with 2x
Gibson Assembly Master Mix (NEB,USA)
3.
Incubate at 50 ̊C for 30 min
4.
Transform up to 5 μl into chemical competent
E. coli
Stel
lar cells via heat shock as described
above