Golden Gate Assembly of RMCE Integration plasmids
Michael Nonet
Department of Neuroscience
Washington University School of Medicine
St. Louis, MO 63110
mnonetWU@gmail.com
9/22/2023 version 2.1.1
1
Extended data for Knoebel, Dour and Nonet, 2023
Table of Contents
I: Overview of RMCE integration vectors
3
II: Overview of Golden Gate plasmid assembly
4
III: In silico assembly of plasmids
7
IV: RMCE integration plasmid assembly
8
Protocol 1: Golden Gate-based RMCE plasmid assembly
8
P1.1 Combining DNAs
8
P1.2 SapI Golden Gate reaction
8
P1.3 General E. coli transformation method
8
V: Creating insert PCR fragments
9
Protocol 2: Creating insert PCR fragments
9
P2.1 Design of oligonucleotides for PCR
9
P2.2 General PCR method
9
P2.3 General column DNA puri
fi
cation method
9
VI: Annealed oligonucleotide pairs as inserts
10
Protocol 3: Annealing oligonucleotides
10
P3.1 Annealing oligonucleotides
10
VII: Creating insert clones
10
Common insert cloning approaches:
10
A) BsaI Golden Gate method
12
Protocol 4: BsaI Golden Gate Method
12
P4.1 Oligonucleotide design
12
P4.2 PCR Ampli
fi
cation of insert
12
P4.3 Combining DNAs
12
P4.4 BsaI Golden Gate reaction
13
P4.5 Transformation into E. coli and analysis
13
B) SapI cloning method
13
Protocol 5: SapI ligation cloning
14
P5.1 Oligonucleotide design
14
P5.2 PCR ampli
fi
cation of insert
14
P5.3 Combining DNAs
14
P5.4 Digest DNA
14
P5.5 Column purify digested DNA
14
P5.6 Ligate DNA
14
P5.7 Transformation into E. coli and analysis
14
C) Gibson assembly method
14
Protocol 6: Gibson Assembly
15
P6.1 Oligonucleotide design
15
Construction of RMCE targeting plasmids
P6.2 PCR ampli
fi
cation of vector and insert
15
P6.3 Gibson assembly reaction
15
P6.4 Transformation into E. coli and analysis
16
D) Double sticky-end cloning
16
Protocol 7: Double sticky-end cloning
17
P7.1 Oligonucleotide design
17
P7.3 Restriction digestions
17
P7.4 Gel puri
fi
cation of DNA fragments
17
P7.5 Ligation
17
P7.6 Transformation into E. coli and analysis
17
E) Multi-fragment BsaI cloning method
17
Protocol 8 Multi-fragment BsaI Golden Gate cloning
18
P8.1 Oligonucleotide design
18
P8.2 Ampli
fi
cation of inserts
19
P8.3 Combining DNAs
19
P8.4 BsaI Golden Gate reaction
19
P8.5 Transformation into E. coli and Analysis
19
F) Multi-Fragment Gibson assembly
20
Protocol 9 Multi-fragment Gibson assembly
20
P9.1 Oligonucleotide design
20
P9.2 PCR ampli
fi
cation of vector and inserts
20
P9.3 Gibson Assembly reaction
20
P9.4 Transformation into E. coli and analysis
20
VIII: Troubleshooting BsaI and SapI Golden Gate cloning
21
References
22
Table 1. DR274 BsaI GG SapI entry cloning vectors
23
Appendix I Reagents
24
TE buffer
24
PE wash buffer
24
PB buffer
24
10X SapI buffer
24
10X Anneal buffer
24
SOC media
24
Appendix II High Fidelity overlap sets from Potapov et al. (2018)
25
Footnotes
26
Construction of RMCE targeting plasmids
2
I: Overview of RMCE integration vectors
Recombination-Mediated Cassette Exchange (RMCE) is a method for creating transgenic animals
using recombinases (Nonet, 2020, 2023). The recombination events catalyze the exchange of DNA between
two distinct FRT sites in a plasmid and similar FRT sites present in specialized landing sites in the genome.
Two distinct approaches have been developed by my lab. The first approach (RMCE) uses a vector
2
containing a self-excising cassette (SEC) that permits both selection for the presence of the cassette as well as
a heat shock controlled Cre recombinase gene to drive self-excision of the cassette after it has been integrated
into the genome. The second approach, named rapid RMCE (rRMCE), uses vectors that contain a fluorescent
protein gene and a selection marker (usually Hyg
R
or Neo
R
) to select for insertions. However, the selection
marker is not excised during the process, but rather, if desired, can subsequently be excised by crossing
through a germline Cre expressing line. Sequences to be integrated can be inserted into the vector by either
traditional restriction enzyme cloning, Gibson assembly or Golden Gate assembly. The vectors for RMCE and
rRMCE are distinct (
Figure 1
), and though both types are technically cross compatible, some of the features of
each method are lost by mixing systems. Thus, choosing the correct vector is an important part of developing
a transgenesis strategy. This manual concentrates on using Golden Gate assembly to create integration
plasmid constructs.
Construction of RMCE targeting plasmids
3
Figure 1 Organization of the pLF3FShC RMCE and pHygG2 rRMCE integration vectors.
A) The pLF3FShC integration vector contains partial Self Excision Cassette (SEC) lacking one
loxP
site. This
consists of a promoter-less hygromycin (
hyg
R
) gene that permits selection of insertions into landing sites, a
sqt-1(e1350)
dominant mutation that permits visual screening for insertions, and a
hsp-16
heat shock promoter driven
Cre
recombinase which permits excision of the selection cassette. Sequences to be integrated are inserted into the
multiple cloning site (MSC) between the
loxP
and
FRT3
site. B) The HygG2 vector contains an MCS, an
FRT
site,
mNG
, a
hyg
R
cassette and an
Amp
R
plasmid backbone, a
loxP
site and an
FRT3
site. Sequences to be integrated are
inserted into the MSC between the
FRT3
and
FRT
sites. See
https://sites.wustl.edu/nonetlab/rmce-integration/
for more
details on how integration works.
sqt-1p
FRT
e1350
SQT-1
sqt-1 3'UTR
tbb-2 3'UTR
Cre
F1 ori
hsp16-41 promoter
7762 SbfI (1)
7782 SphI (1)
7797 XmaI (1)
pLF3FShC [3643]
7855 bp
AmpR
hygR
7756 SapI (2)
7786 SapI (2)
FRT3
7747 SpeI (1)
loxP
7768 BstEII (1)
7775 MluI (1)
ColE1 origin
sqt-1 5'UTR
let-858 Terminator
myo-2 3' UTR
SV40 nls
FRT
unc-54 3'UTR
hygR
ColE1 origin
Prps-0
AmpR
F1 ori
loxP
5907 NotI (1)
5921 SpeI (1)
5930 SapI (2)
5936 SbfI (1)
5942 BstEII (1)
5956 SphI (1)
5960 SapI (2)
5967 KpnI (1)
5971 XmaI (1)
FRT3
pHygG2 [4332]
6029 bp
Synthetic Intron 1
egl-13 NLS
Synthetic Intron 4
Synthetic Intron 3
NeonGreen_Cel
Synthetic Intron 2
II: Overview of Golden Gate plasmid assembly
The Golden Gate assembly (Engler et al. 2008) method facilitates simultaneously inserting multiple
DNA fragments that each contain an element to be included in the final clone. Each element is flanked by
restriction sites, which end up excluded from the final assembly. The method takes advantage of type IIS
restriction enzymes, such as
SapI
, that cut at a non-palindromic recognition sequence and cleave the DNA
outside its restriction selectivity site and leave a 5’ overhang cohesive end. For example,
SapI
cleaves at 5’
GCTCTTCN/NNN 3’. This it leaves a 3 base overhang that can be any sequence. By assembling fragments
with different overhangs, one can assemble multiple fragments, and these will assemble only in a specific
order based on base pairing (
Figure 2
).
Since the restriction enzyme sites are excluded from the final assembly, the digestion and ligation steps
can be repeated numerous times to increase the efficiency of the assembly process. Using the Golden Gate
method, it is possible to assemble over 10 fragments into a vector without too much difficulty using high quality
DNA reagents. Although in theory any order of 3 base overhangs can be used to create plasmids, I use a
specific set of pairs of overhangs as ‘slots’ and use a conventional order to facilitate the sharing of reagents.
This permits development of libraries of clones which can be used interchangeably in the construction of new
integration plasmids. An additional advantage of using sequenced plasmids as the source of inserts is that the
final assembly does not require sequencing. Alternatively, the inserts can be supplied as PCR fragments.
Very small inserts can be supplied as hybridized oligonucleotide pairs.
Construction of RMCE targeting plasmids
4
Figure 2. Golden Gate Cloning strategy using SapI 3 bp overlaps.
Examples of Golden Gate assembly using either plasmids or PCR fragments as the source of inserts. S presents a
SapI
restriction site. The plasmid backbone of the vector (brown) is Amp
R
of the inserts (black) are Kan
R
.
S
S
S
S
S
S
S
S
S
S
S
S
GTA
TGG
S
S
GTA
TGG
S
S
TGG
GCG
GCG
ATG
ATG
AAG
GGT
GGT
ACG
ACG
GTA
AAG
S
S
S
S
S
S
S
S
S
S
S
S
mix
ligate
10
-
30 X
SapI
digest
mix
ligate
10
-
30 X
SapI
digest
To assemble integration constructs I use a
SapI
Golden Gate strategy based on Dickinson's method for
building CRISPR integration plasmids (Dickinson et al. 2018), which is a merging of two approaches, one
developed by Dickinson et al. (2015) and one by Schwartz and Jorgensen (2016). The basic methodology
(see below) involves introducing up to 8 distinct DNA fragments in order into an RMCE integration vector.
Each element has distinct
SapI
sites at its ends forcing the ordered assembly (
Figure 2
).
For historical reasons these are often refer to these slots as the ’sgRNA’, ’pU6’, ‘5’ arm’, ‘CT,’ ‘FP’,
‘SEC’, ‘NT’ and '3’ arm’ slots because originally this approach was used to created SEC containing CRISPR/
cas9 integration vectors (Dickinson et al., 2018). However, in our approach the SEC has been incorporated
into the vector rather than being introduced as one of the inserts.
The approach is powerful for several reasons. First, many traditional cloning steps are combined into
one using this approach. Second, as mentioned above, the approach permits one to use previously
constructed plasmids as inserts which allows for developing “libraries” of inserts which facilities rapid
construction of new integration constructs. The one major limitation is that these inserts cannot contain a
SapI
restriction site. If
SapI
sites are present in a potential insert they must be eliminated. The manual discusses
how to remove sites if they are present.
In addition to pLF3FShC and pHygG2 (and related vectors) which are designed for six slots, pLF3FShC2 and
pHygG12 (and related vectors) are designed for 8 slots with two additional slots on the 5’ end (relative to
pLF3FShC and pHygG2). Although I define these as ‘six slot’ and ‘eight slot’ vectors, I often merge slots when
constructing simpler insertions (
Figure 3
). In theory one can also divide slots. The number of fragments one
can insert is technically only limited by the number of different 3 bp overhangs. This approach provides great
flexibility in creating new constructs. For example, a very common construct one would create is an N-terminal
fluorescent protein (FP) fusion to a protein under a specific promoter. In this case, I place our promoters is a
combined 5’ arm-CT slot vector, use the FP slot for a fluorescent protein the SEC slot for a linker, the NT slot
for our gene of interest, and the 3’ arm slot for the 3’ UTR of the construct (
Figure 3
). One can switch the
promoter to create a construct that will express the fusion in a different cell type, the FP to express a different
color fusion, or the gene of interest to make a distinct FP-protein fusion. As one has builds up libraries of
promoters, FPs and cloned genes creating new derivatives is becomes simpler and simpler.
Construction of RMCE targeting plasmids
5
Construction of RMCE targeting plasmids
6
Figure 3. Cloned insert libraries provide the flexibility to quickly assemble novel constructs.
Examples of various integration clones that can be assembled from SapI entry clone libraries (Knoebel et al.
2023) in combination with a gene of interest PCR product. Common promoters, FPs, tags and 3’ UTRs are supplied
from the clone library, other small tags can also be supplied as hybridized oligonucleotide pairs (not shown) and the
gene ORF is supplied as a PCR product flanked with
SapI
restriction sites. S presents a
SapI
restriction site. The
plasmid backbone of the vector (brown) is Amp
R
of the inserts (black) are Kan
R
.
S
S
S
S
S
S
GTA
TGG
S
S
promoter
FP or ...
3’ UTR
your gene
promoter library
fluorescent protein libraries
3’ UTR libraries
S
S
S
S
S
S
promoter
FP
3’ UTR
S
S
linker
S
S
S
S
S
S
promoter
FP
3’ UTR
S
S
linker
your gene
S
S
S
S
S
S
S
S
tag
S
S
promoter
tag
sl2
FP
3’ UTR
ATG
AAG
S
S
GGT
ACG
S
S
ATG
AAG
S
S
your gene
'linker' library
tag library
III:
In silico
assembly of plasmids
The plasmid editor ApE (Davis and Jorgensen, 2022) facilitates design and assembly of plasmids. It
provides a tool to assemble plasmids using Golden Gate reactions i
n silico
. To use the tool, create ApE files
representing each PCR product, insert plasmid, and the plasmid vector. Unfortunately, the program does not
deal well with oligonucleotide pairs. One needs to create a file that represents the oligonucleotide pair with
SapI
sites on the ends. To create a plasmid (
Figure 4
), open the vector file and all the insert files. Select Tool/
Golden Gate Assembler, then select the enzyme being used for the reaction. Finally select all of the inserts
and click OK. The program will create a new file of the fully assembled product. If ‘No Circular Product
Possible” appears then one of the inserts is missing or not properly designed. Using this program to test
designs will save one from silly mistakes in choosing plasmids, oligonucleotides for amplifying products, etc.
A second tool available for assembling RMCE plasmids that provides a better overview of the potential
to create clones but provides less well annotated plasmids is an Excel based assembly tool called ‘GG
assembly Builder’ available at
https://sites.wustl.edu/nonetlab/golden-gate-cloning-resources/#GGABuilder
.
This excel document (
Figure 5
) has a series of dropdown menus which display the clones available from the
Nonet lab (and some compatible plasmids published by other labs) which can be used to create assembles in
either 6 or 8 slot vectors. One selects the vector, and the desired inserts for each slot, and the tool builds both
a full sequence and an annotated sequence file. The contents can be pasted into a text document to create a
sequence file or Genbank format annotated file.
Construction of RMCE targeting plasmids
7
Figure 5. GG Assembly Builder
in silico
assembly tool
An example assembly of a pHygG2
glr-1p::
TIR1-V2A-Scarlet::tbb-2 3’ UTR rRMCE integration vector.
Figure 4. ApE in silico Golden Gate Reactions.
Assembly of a tetO 7Xp::mNG-linker-rab-3::tbb-2 3’ UTR construct in pHygG2. Left) a functional and right) a non-
functional assembly.
IV: RMCE integration plasmid assembly
Building a new RMCE integration vector involves
1. Designing the new construct.
2. Creating the novel PCR fragments or clones required for the final assembly.
3. Performing a
SapI
Golden Gate reaction to build the construct using the DNAs.
I address these steps in reverse order since it is possible to acquire many insert containing plasmids for
assembling novel plasmids (from Addgene, the Nonet lab and by scouring publications) without performing any
new cloning or PCR fragment amplification. I do not discuss design of integration plasmids as this is a
complicated issue. A extensive discussion of clone design is found in Nance and Frøkjær-Jensen (2019).
Protocol 1: Golden Gate-based RMCE plasmid assembly
P1.1 Combining DNAs
3
Clones, PCR products, and annealed oligonucleotides are mixed using the following ratios
4
20 fmol of vector (~75 ng of the 6 kb pHygG2 vector)
20 fmol of each insert (e.g. 50 ng for a 4 kb plasmid, 12 ng of 1kb PCR product)
50 fmol of each oligonucleotide pair (e.g. 2 ng for a 60 bp oligonucleotide pair)
Dilute to 10
μ
l with TE if volume is less than 10
μ
l.
P1.2
SapI
Golden Gate reaction
1
μ
l 10X Sap reaction buffer
5
7
μ
l H
2
0
1
μ
l DNA mix
1/2
μ
l of SapI
6
1/4
μ
l of T4 DNA ligase
1/4
μ
l of Polynucleotide Kinase (optional
)
7
Perform the digestion/ligation in a PCR machine
. Typically, I run 37° for 10 min, followed by 10 cycles of
8
(16°C for 5 min, 37° for 5 min), then a step of 65°C for 20 min. The reaction is even more efficient if one uses
30 cycles (which I do if I am running the reaction overnight). The last 37°C step is to allow
SapI
to do a last
round of cutting of incomplete assemblies, so they are not circles. The 65°C step inactivates the enzymes.
P1.3 General
E. coli
transformation method
Thaw a tube of competent cells
on ice for 10-15 minutes.
9
Aliquot 50-100
μ
l to a pre-chilled tube on ice.
Add 1
μ
l of ligation to the competent cells and briefly vortex.
Incubate on ice 20-40 minutes (longer the better).
Heat shock 30 sec at 42°C.
Incubate 10-20 minutes on ice (longer the better).
Add 700
μ
l of SOC media.
Incubate 1 hr at 37 with shaking.
Plate 1/10
th
of the transformation on the appropriate drug
LB plate, and incubate o.n. at 37°C.
10
One should get hundreds of colonies using competent cells with 1 x 10
8
colonies/
μ
g pBluescript DNA
efficiency. A minimum of ~30-80% of colonies are correct in my experience. Often it is 100%.
P1.4 Clone analysis
Miniprep
3-6 colonies and identify putative correct clones using a restriction digest (
EcoRI
is usually
11
diagnostic). Perform additional restriction digests to confirm all inserts are present if small inserts (linkers, V2A
tags) could be absent without significantly altering the digestion product pattern. If the inserts are derived from
plasmids that have already been sequence verified, then I find sequencing is not needed. If the inserts are
PCR products the error rate will depend on the quality of the template used and the error rate of the
polymerase. It is rare to find errors using quality genomic DNA as template and NEB Q5 as the polymerase.
Construction of RMCE targeting plasmids
8
V: Creating insert PCR fragments
One approach for creating inserts is to PCR amplify the desired fragments from a high-quality source of
DNA (genomic DNA, first strand cDNA, or a plasmid). However, if the fragment of interest contains
SapI
sites,
they need to be ‘removed’ by introducing silent mutations. In such cases I find it is easier to remove the sites
using a
BsaI
Golden Gate multi-insert strategy (see below) while cloning the fragment into a Kan
R
plasmid.
Alternatively, if multiple
SapI
sites are present, it may be simpler to have the fragment synthesized
in vitro
, or to
perform the assembly using alternative methods such as Gibson assembly
.
12
Protocol 2: Creating insert PCR fragments
P2.1 Design of oligonucleotides for PCR
Design appropriate oligonucleotides that will amplify the product of interest. Several things need to be
checked. First the fragment cannot have any
SapI
sites in it. The 5’ ends of each primer should contain a
SapI
site and the appropriate 3 bp overlap for the slot one is inserting into as shown in Figures 2 & 3. For example,
for the fragment in the 5’ arm slot they should be 5’ GACT
GCTCTTC
gTGG and 5‘ CACT
GCTCTTC
gCGC. The
first four bases are to ensure efficient cutting by
SapI
. If the construct being assembled creates a protein
13
fusion, remember to maintain frame at junctions between your insert sequences.
P2.2 General PCR method
Standard 25 ul PCR reaction
In one PCR tube
on ice
:
16
μ
l H20
5
μ
l of 5X Q5 polymerase buffer
2
μ
l 2.5 mM dNTPs
1
μ
l template DNA (25 ng N2 genomic DNA or 1ng of plasmid)
14
1/2
μ
l 10-25 uM forward oligonucleotide
15
1/2
μ
l 10-25 uM reverse oligonucleotide
To reaction mix add 1/4
μ
l of Q5 polymerase
. Mix well by pipetting and return to ice.
16
Start PCR machine using an appropriate program and add tube(s) when machine hits 72°C
.
17
Typical PCR conditions for oligonucleotides designed with a 55-58°C annealing temperature
:
18
98°C 0:30, 30 cycles (98°C for 0:10, 58-62°C for 0:30, 72°C for 1:00/kb).
Optional: (required if the template is an Amp
R
plasmid) Add 1/2
μ
l of
DpnI
and digest 15 minutes at 37°C.
P2.3 General column DNA puri
fi
cation method
Add 125
μ
l (5 volumes) of diluted PBI
to the PCR reaction.
19
Load the reaction on a Qiagen or Monarch mini column.
20
Spin 30 seconds to bind to column (optional - remove binding liquid).
Add 150
μ
l PE buffer, spin 30 sec. (optional- remove PE).
Add 200
μ
l PE buffer, spin 30 sec. Rotate tube 180 degrees, Spin 1 minute.
Transfer column to a 1.5 ml Eppendorf tube.
Add 10
μ
l of TE. Spin at 100 RCF, 1 minute to drive TE into column.
Spin full speed 1 minute.
(optional) To increase yield, but lower concentration, add 10
μ
l TE and repeat elution.
A typical yield will be ~1-2
μ
g of DNA for a 25
μ
l PCR reaction
.
21
Quantify using a NanoDrop spectrophotometer, or more crudely by running a 1
μ
l aliquot on a gel with a known
concentration of a DNA ladder.
Construction of RMCE targeting plasmids
9
VI: Annealed oligonucleotide pairs as inserts
In cases where one would like to insert a very small fragment in one of the slots (for example adding a
FLAG or HA tag to the end of a FP insert, one can use a pair of oligonucleotides that have been annealed as a
fragment in the
SapI
assembly reaction. The key here is to make sure that one does not add too much
fragment into the reaction by accounting for the small size of the oligonucleotide fragment. For the typical 60
bp annealed insert, 1 ng of each oligonucleotide would be ~ 50 fmol.
Protocol 3: Annealing oligonucleotides
P3.1 Annealing oligonucleotides
Mix 1
μ
l of each 100 uM oligonucleotide stock (100 pmol/
μ
l) in 10
μ
l of water
Add 1
μ
l of that dilution to 100
μ
l 1X anneal buffer
Heat to 95°C for 2 minutes and slow cool to 37 at -6°C per minute in a PCR machine.
This creates a working stock at 100 fmol/
μ
l of the annealed oligonucleotides. Remember to add PNK to the
Golden Gate reaction when using oligonucleotides as these are not phosphorylated on the 5’ end.
VII: Creating insert clones
Another option is to create a
SapI
entry clones for the fragment(s) instead of using a PCR product.
This may save time if one is going to use the insert for multiple different integration constructs. In particular,
once the plasmid has been sequenced, one can be confident the final product will not have an error, while if
the insert is a PCR product errors are more likely. The general approach is to PCR amplify the product of
interest, digest it with restriction enzymes, and ligate the fragment into a Kan
R
vector. Several approaches can
be used which are outlined in
Figure 6
.
Common insert cloning approaches:
There are many approaches that one can use to create
SapI
insert clones. The ones that the Nonet lab
uses most often are:
A)
BsaI
Golden Gate method
In this approach the insert fragment is introduced into a plasmid that has the
SapI
sites using a
BsaI
Golden Gate cloning strategy. This is the simplest approach if your insert does not contain
BsaI
sites. It is
virtually 100% reliable, rapid and requires very little DNA. Specifically, I have created Kan
R
vectors for each
slot (e.g. the ‘FP’ ATG AAG slot), that permits inserting the fragment by adding
BsaI
sites on the end of the
fragment. This ‘only’
works if the fragment does not contain
BsaI
sites, but it is very easy to perform.
22
B)
SapI
ligation method
In this approach the
SapI
fragment is introduced into the same vector described in A) but using a
SapI
digestion and ligation. This approach is used for inserts that contain
BsaI
sites or to clone a fragment that
originally was inserted as a PCR fragment.
C) Gibson assembly method
Gibson assembly (Gibson, 2011) is a popular approach of cloning DNA fragments and utilizes 15-20 bp
of homology between the insert and the vector to drive the specificity of the reaction. This is alternative
method to insert fragments that contain
BsaI
sites.
D) Double sticky-end cloning method
Another well-vetted approach to clone fragments which relies on double sticky-end restrictions sites to
drive orientation and specificity of the cloning reaction into a Kan
R
(or other non-Amp
R
vector).
E) Multi-fragment
BsaI
Golden Gate method
In cases where there are
SapI
(or
BsaI
) sites in the fragment of interest and one wishes to clone the
fragment, one can use a multi-insert
BsaI
Golden Gate strategy to lesion all the sites (using synonymous
changes if in coding sequence) and assemble a
BsaI
and
SapI
lacking version of the insert.
F) Multi-fragment Gibson assembly method
A similar strategy to E) can also be implemented using Gibson assembly driving proper assembly by
using homology rather than restriction endonucleases. This is particularly useful for fragments that contain
many
BsaI
sites in addition to
SapI
sites.
Construction of RMCE targeting plasmids
10
Construction of RMCE targeting plasmids
11
Figure 6. Alternatives for cloning inserts into Golden Gate ‘entry’ vectors.
Six alternative methods for creating Golden Gate entry vectors. A) a
BsaI
Golden Gate approach, B) a
SapI
cloning approach, C) a Gibson assembly cloning approach, D) a traditional double sticky-end cloning approach E) a
multi-fragment
BsaI
Golden Gate approach, and F) a multi-fragment Gibson assembly cloning approach. Restrictions
sites: B=
BsaI,
R=
EcoRI
, H=
HindIII
, S=
SapI
. Inserts are shown in red and the vector in black.
B
B
S
S
B
B
B
S
S
B
mix
BsaI
digest
ligate
transform
S
S
10 X
S
S
B
B
S
S
mix
SapI
digest
purify
ligate &
transform
S
S
PCR
PCR
A
B
C
S
S
Hi
-
Fi
reaction
50
°
C , 1hr
transform
S
S
mix
PCR
B
B
S
S
v
i
S
H
R
PCR
S
H
R
EcoRI, HindIII
digest
ligate &
transform
S
S
D
gel purify
B
S
S
B
BsaI
digest
ligate
transform
10 X
mix
B
*
S
*
B
B
B
B
B
B
PCR
S
S
E
Hi
-
Fi
reaction
50
°
C , 1hr
transform
mix
PCR
F
B
*
S
*
S
S
S
S
A)
BsaI
Golden Gate method
This simple method works highly efficiently and is our favorite approach for inserts that contain neither
a
BsaI
or a
SapI
site. One amplifies the insert of interest with oligonucleotides that append
BsaI
sites on each
end of the product and insert the fragments into the appropriate DR274 slot vector. Vectors are available for all
eight slots and some common combined slots (
Ta b l e 1
). An example is shown in
Figure 7
.
Protocol 4:
BsaI
Golden Gate Method
P4.1 Oligonucleotide design
Design oligonucleotides with
BsaI
sites each designed to match the overlap of the appropriate DR274
slot-
BsaI
vector (
Ta b l e 1
). For example, for the FP vector, the overlaps are GATG on the 5’ side and AAGG for
the 3’ side. Thus, the oligonucleotides should start with 5’ AGGTCTCAGATG 3’ and 5’ AGGTCTCACCTT 3’. A
single base 5’ of the
BsaI
site is added to ensure this PCR fragments cut effectively with
BsaI
. Remember to
make sure that the correct reading frame is maintained if one is cloning an ORF or functional domain.
P4.2 PCR Ampli
fi
cation of insert
Amplify and purify the PCR product using the
general PCR method
and purify using the
general column
purification method
.
P4.3 Combining DNAs
Mix
50 fmol of plasmid DR274 BsaI-Slot (50 ng vector)
60 fmol Insert PCR (25 ng of 1 kb insert)
Add TE to 10
μ
l of TE total
Construction of RMCE targeting plasmids
12
Figure 7. BsaI
Golden Gate insert cloning into DR274 slot entry vectors.
Schematic diagram of
BsaI
Golden Gate cloning to create
SapI
Golden Gate entry clones. Shown is an insert PCR
product with
BsaI
sites on each end. The insert and vector are mixed and co-assembled using a
BsaI
Golden Gate
reaction. The limitation of this method is that the insert cannot contain either a
BsaI
site or a
SapI
site. Red arrowheads
represent the cut sites for
SapI
and orange arrowheads represent the cut sites for
BsaI.
SapI
BsaI
BsaI
SapI
| | | |
-
GAATTC
GCTCTTC
GATGA
GAGACC
gcaatGGATCCaaccatt
GGTCTC
AAAGG
GAAGAGC
AAGCTT
-
-
CTTAAG
CGAGAAG
CTACT
CTCTGG
cgttaCCTAGGttggtaa
CCAGAG
TTTCC
CTTCTCG
TTCGAA
-
SapI
19 BsaI 43 BsaI
SapI
|
| |
|
-
GAATTC
GCTCTTC
GATGNNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNAAGG
GAAGAGC
AAGCTT
-
-
CTTAAG
CGAGAAG
CTACNNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNTTCC
CTTCTCG
TTCGAA
-
BsaI
BsaI
| |
a
GGTCTC
AGATGNNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNAAGGA
GAGACC
g
t
CCAGAG
TCTACNNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNTTCCT
CTCTGG
c
aGGTCTCA
GATGNNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNN
AAGGAGAGACCg
tCCAGAGTCTAC
NNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNTTCC
TCTCTGGc
SapI
19 BsaI 43 BsaI
SapI
|
| |
|
-
GAATTC
GCTCTTC
gATGAGAGACCgcaatGGATCCaaccattGGTCTCA
AAGG
GAAGAGC
AAGCTT
-
-
CTTAAG
CGAGAAG
CTAC
TCTCTGGcgttaCCTAGGttggtaaCCAGAGTTTCc
CTTCTCG
TTCGAA
-
Vector
Insert
Product
BsaI
digest
BsaI
digest
Ligate
Ligate
BsaI
:
5’ NNN
GGTCTC
N 3’
3’ NNN
CCAGAG
NNNNN 5’
SapI
:
5’ NNN
GCTCTTC
N 3’
3’ NNN
CGAGAAG
NNNN 5’
VECTOR
P4.4
BsaI
Golden Gate reaction
1
μ
l of 10 Sap reaction buffer
1/2
μ
l of DNA mix
7
μ
l H20
1/2
μ
l
BsaI
1/4
μ
l T4 DNA ligase
Run reaction: 37°C, 10 min; 16°C, 5 min; 10X (37°C, 2 min; 16°C, 2 min); 37°C,10 min; 65°C 10 min.
23
P4.5 Transformation into
E. coli
and analysis
Transform using the general
E. coli
transformation method
with 1/2
μ
l and plate 1/10
th
of the
transformation. This should yield thousands of colonies and virtually all will be correct. Analysis of 3 clones
should be sufficient to get a clone and a backup (in case of PCR induced error). Sequencing of clones is wise
for those encoding ORFs and recommended for those encoding promoters, intergenic regions, or 3’ UTRs.
B)
SapI
cloning method
I use this approach if there are internal
BsaI
sites in the fragment I am attempting to clone and I have
chosen not to remove them. An example is shown in
Figure 8
.
Construction of RMCE targeting plasmids
13
Figure 8.
SapI
insert cloning into DR274 slot entry vectors.
Schematic diagram of
SapI
restriction enzyme cloning to create
SapI
GG entry clones. Shown is an insert PCR
product with
SapI
sites appended on each end. The insert and vector are mixed, digested with
SapI
, column puri
fi
ed
and assembled using a ligation reaction. Red arrowheads represent the cut sites for
SapI.
SapI
BsaI BsaI
SapI
|
|
|
|
-
GAATTC
GCTCTTC
GATGAGAGACCgcaatGGATCCaaccattGGTCTCAAAGG
GAAGAGC
AAGCTT
-
-
CTTAAG
CGAGAAG
CTACTCTCTGGcgttaCCTAGGttggtaaCCAGAGTTTCC
CTTCTCG
TTCGAA
-
SapI
SapI
| |
GACT
GCTCTTC
AATGNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNNAAGG
GAAGAGC
AGTG
CTGA
CGAGAAG
TTACNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNNTTCC
CTTCTCG
TCAC
SapI
BsaI BsaI
SapI
|
|
|
|
-
GAATTC
GCTCTTC
GATGNNNNNNNNNN
--
INSERT
—
NNNNNNNNNNNNNAAGG
GAAGAGC
AAGCTT
-
-
CTTAAG
CGAGAAG
CTACNNNNNNNNNN
--
INSERT
—
NNNNNNNNNNNNNTTCC
CTTCTCG
TTCGAA
-
SapI
SapI
| |
TTGCTCTTCA
ATGNNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNN
AAGGGAAGAGCAA
AACGAGAAGTTAC
NNNNNNNNNN
--
INSERT
--
NNNNNNNNNNNNNTTC
CCTTCTCGTT
SapI
digest
SapI
digest
Ligate
Ligate
SapI
BsaI saI
SapI
|
|
|
|
-
GAATTC
GCTCTTC
G
ATGAGAGACCgcaatGGATCCaaccattGGTCTCA
AAGG
GAAGAGC
AAGCTT
-
-
CTTAAG
CGAGAAG
CTAC
TCTCTGGcgttaCCTAGGttggtaaCCAGAGTTTC
C
CTTCTCG
TTCGAA
-
VECTOR
Protocol 5:
SapI
ligation cloning
P5.1 Oligonucleotide design
Design appropriate oligonucleotides that will amplify the product of interest. The 5’ ends of each primer
should contain a
SapI
site and the appropriate 3 bp overlap for the slot vector one is utilizing. For example, for
the fragment being introduced in the 5’ arm slot they should be 5’ GACT
GCTCTTC
gTGG and
5‘ CACT
GCTCTTC
gCGC. Add four bases 5’ of the
SapI
recognition site to ensure efficient cutting by
SapI
. If
the construct being assembled creates a protein fusion, remember to maintain frame at junctions between your
insert sequences.
P5.2 PCR ampli
fi
cation of insert
Amplify the fragment using the
general PCR method
and purify using the
general column purification
method
.
P5.3 Combining DNAs
Mix
50 fmol of plasmid DR274
BsaI
slot (50 ng vector)
60 fmol insert PCR (25 ng of 1 kb insert)
dilute to 10
μ
l with TE
P5.4 Digest DNA
7
μ
l H2O
1
μ
l 10X NEB Smart buffer
2
μ
l DNA mix
1/3
μ
l of
SapI
Incubate 30 min to 1 hr.
P5.5 Column purify digested DNA
Dilute the digestion to 25 ul and column purify as outlined in
purification of PCR product
.
This step is
performed to remove the small
SapI
insert of the vector which greatly increases the efficiency of the ligation by
eliminating competition for reinsertion that fragment into the vector. It is important to use diluted PBI otherwise
the insert will stay on the column and the purification will accomplish little.
P5.6 Ligate DNA
To the eluted DNA (20 ul), add 2ul 10X T4 DNA ligase buffer and 1/4
μ
l of T4 DNA ligase. Ligate 30 minutes.
P5.7 Transformation into
E. coli
and analysis
Transform using the general
E. coli
transformation method
with 1/2
μ
l of ligation mix and plate 1/10th of
transformation. You should get thousands of colonies and the majority will be correct insert clones. Analysis of
3 clones should be sufficient to get a clone and a backup (in case of PCR induced error). Sequencing of
clones is necessary for those encoding ORFs and recommended for those encoding promoters, intergenic
regions or 3’ UTRs.
C) Gibson assembly method
DR274 slot vectors do not exist for all combinations of
SapI
sites. In some cases, one may use an
unusually combination because one is adding a slot or deleting a slot to create an unusual plasmid. In these
cases, cloning the PCR fragments to create an entry vector is easiest done by either Gibson assembly or
standard double sticky-end cloning. In either case, the
SapI
sites with appropriate ends and either 15 bp of
homologous sequences or restriction sites are appended to the 5’ end of the oligonucleotides to create
appropriate PCR products. This can be used both to create clones using single inserts or more complex
assemblies using multiple fragments (to alter internal
BsaI
or
SapI
sites). A major benefit of Gibson assembly
over Golden Gate cloning is that there is footprint left at the junction while in our Golden Gate strategy there is
a 3 bp footprint. Gibson assembly is also relatively insensitive to the presence of primer dimers unlike the
Golden Gate approach. Gibson Assembly uses homology between 15-30 bp overlaps between DNA fragments
(usually created by PCR) to drive the proper assembly. Any source or DNA (gel purified restriction digestion
Construction of RMCE targeting plasmids
14
products, synthetic DNA, or PCR products can be used as the substrate for a Gibson assembly reaction).
However, we have found in the past that PCR is the most efficient approach as restriction digested vectors
sometimes contain a very small amount of partially cut product that can cause issues with background. We
use the Hi-Fi 2X master mix from NEB to perform our reactions. To create a novel clone using this strategy
oligonucleotides are designed to create vector and insert fragments that have 15-20 bp overlaps - longer
overlaps work better. The vector and insert fragments are PCR amplified,
DpnI
digested to remove template
DNA, and purified. The fragments are then mixed with 2X NEB Hi-Fi master mix for 1 hr. and transformed.
The approach is efficient but requires more DNA that Golden Gate cloning. An example is shown in
Figure 9
.
A note of caution about this protocol. The Nonet lab no longer uses Gibson assembly very often and thus I
have not optimized this protocol and I do not keep up on ‘advances’ in Gibson assembly strategies.
Protocol 6: Gibson Assembly
P6.1 Oligonucleotide design
To create a clone by Gibson assembly I typically create the clone
in silico
by cutting and pasting the
insert(s) into the vector of choice to create an
in silico
version which marks all fragment junctions. I then
decide which oligonucleotide should be appended to add the overlap. This determines which purified PCR
fragments are easily re-usable for other Gibson assembly reactions. A 55°C T
m
overlap is our typical choice.
The desktop program ApE and the NEBuilder website both have tools to facilitate the design of primers for
Gibson assembly. An example is shown in
Figure 9
.
P6.2 PCR ampli
fi
cation of vector and insert
Amplify the vector and insert using the
general PCR method
and purify using the
general column purification
method
.
P6.3 Gibson assembly reaction
Mix the following
25 fmol of vector PCR
Construction of RMCE targeting plasmids
15
Figure 9 Gibson Assembly.
Schematic diagram of Gibson assembly cloning to create
SapI
GG entry clones. Shown is an insert PCR
product with
SapI
sites and a small homology arm appended on each end. The insert and vector are mixed and co-
assembled using a HiFi Gibson assembly reaction. Red arrowheads represent the cut sites for
SapI.
SapI
19 BsaI 43 BsaI
SapI
|
| |
|
-
TTTGAATTC
GCTCTTC
GATGGCAGGCCTG
--
INSERT
--
ACGCGGAGTGCCTAAGG
GAAGAGC
AAGCTTGGAT
-
-
AAACTTAAG
CGAGAAG
CTACCGTCCGGAC
--
INSERT
--
TGCGCCTCACGGATTCC
CTTCTCG
TTCGAAGGTA
-
SapI
19 BsaI 43 BsaI
SapI
|
| |
|
—
TTTGAATTC
GCTCTTC
G
G
GAAGAGC
AAGCTTGGAT
-
-
AAACTTAAG
CGAGAAG
C
C
CTTCTCG
TTCGAAGGTA
-
SapI
SapI
| |
-
TTTGAATTC
GCTCTTC
GATGAGAGACCgcaatGGATCCaaccattGGTCTCAAAGG
GAAGAGC
AAGCTTGGAT
-
-
AAACTTAAG
CGAGAAG
CTACTCTCTGGcgttaCCTAGGttggtaaCCAGAGTTTCC
CTTCTCG
TTCGAACCTA
-
ATGGCAGGCCTG
--
INSERT
--
AGACGGAGTGCCTAAG
TACCGTCCGGAC
--
INSERT
--
TCTGCCTCACGGATTC
PCR
Gibson assembly
Vector
Product
Clone
Template
Vector
PCR
VECTOR
Insert PCR
products
Gibson assembly
SapI
SapI
| |
TTTGAATTC
GCTCTTC
G
ATGGCAGGCCTG
--
INSERT
--
AGACGGAGTGCCTAAG
G
GAAGAGC
AAGCTTGGAT
AAACTTAAG
CGAGAAG
C
TACCGTCCGGAC
--
INSERT
--
TCTGCCTCACGGATTC
C
CTTCTCG
TTCGAACCTA
PCR added
overlap
50 fmol of insert PCR
24
TE to 5 ul
5 ul of 2X HiFi Assembly Master Mix
Incubate at 50°C for 1 hr.
P6.4 Transformation into
E. coli
and analysis
Transform using the general
E. coli
transformation method
with 1/2
μ
l of ligation mix and plate 1/10th of
transformation. The number of colonies obtained is highly dependent on the length of the overlap, but a few
hundred is common for 55°C T
m
overlaps. The majority will be correct insert clones. Analysis of 3 clones
should be sufficient to get a clone and a backup (in case of PCR induced error).
D) Double sticky-end cloning
This is the traditional approach to cloning my lab used to create most single insert clone for most of the
30 years my lab had been doing molecular biology before switch to using Golden Gate cloning for most
applications. It can be more efficient to use that the
SapI
method to clone fragments that contain
BsaI
sites if
one if performing lots of different reactions with different
SapI
overhangs. This is because the
SapI
sites are
incorporated into the PCR insert and thus in theory all the different fragments can be cloned into the same
double cut vector
.
My lab typically uses typically use the plasmid DR274 TbLCTb. It contains the following sites.
HindIII
/
BspHI
/
XbaI
/
SphI
— [1.5 Kb insert w/
HindIII
and
BsrGI
sites] —
EagI
/
BsrGI
/
EcoRI
Construction of RMCE targeting plasmids
16
Figure 10 Double sticky-end cloning.
In this approach a DNA fragment is ampli
fi
ed with oligonucleotides which append both the
SapI
site and
another restriction site on each end of the fragment. The PCR products are then digested, gel puri
fi
ed and ligated
into gel puri
fi
ed digested vector. This approach can simplify the parallel cloning of many inserts with different SapI
overhangs by using common other sites (
EcoRI
and
HindIII
in this example) to perform all the cloning steps. Red
arrowheads represent cut sites for
SapI.
SapI
19 BsaI 43 BsaI
SapI
|
| |
|
-
TTTTGAATTC
GCTCTTC
GATGGCAGGCCTG
--
INSERT
--
ACGCGGAGTGCCTAAGG
GAAGAGC
AAGCTTGGAT
-
-
AAAACTTAAG
CGAGAAG
CTACCGTCCGGAC
--
INSERT
--
TGCGCCTCACGGATTCC
CTTCTCG
TTCGAAGGTA
-
19 BsaI 43 BsaI
| |
—
TTTTG
AGCTTGGAT
-
-
AAAACTTAA
AGGTA
-
EcoRI
HindIII
| |
-
TTTT
GAATTC
GCTCTTCGATGAGAGACCgcaatGGATCCaaccattGGTCTCAAAGGGAAGAGC
AAGCTT
GGAT
-
-
AAAA
CTTAAG
CGAGAAGCTACTCTCTGGcgttaCCTAGGttggtaaCCAGAGTTTCCCTTCTCG
TTCGAA
CCTA
-
ATGGCAGGCCTGACG
--
INSERT
--
AGACGGAGTGCCTAAG
TACCGTCCGGACTGC
--
INSERT
--
TCTGCCTCACGGATTC
Digest
Ligate
Vector
Product
Clone
Te m p l a t e
Digested
vector
VECTOR
PCR
product
Ligate
EcoRI
SapI
SapI
HindIII
| | | |
TTTT
GAATTC
GCTCTTC
GATGGCAGGCCTG
--
INSERT
--
AGACGGAGTGCCTAAGG
GAAGAGC
AAGCTT
GGAT
AAAA
CTTAAG
CGAGAAG
CTACCGTCCGGAC
--
INSERT
--
TCTGCCTCACGGATTCC
CTTCTCG
TTCGAA
CCTA
PCR adding RE sites
EcoRI
SapI
SapI
HindIII
| | | |
AATTC
GCTCTTC
GATGGCAGGCCTG
--
INSERT
--
AGACGGAGTGCCTAAGG
GAAGAGC
A
G
CGAGAAG
CTACCGTCCGGAC
--
INSERT
--
TCTGCCTCACGGATTCC
CTTCTCG
TTCGA
Digest
Insert
Thus, using any combination of 1 of the 4 sites on the left and 1 of the 3 sites on the right of the insertion site
will allow you insert a clone. An example of double sticky-end cloning is shown in
Figure 10
.
Protocol 7: Double sticky-end cloning
P7.1 Oligonucleotide design
Design a pair of oligonucleotides that append the proper overlap (a
SapI
site and an appropriate
cloning site) to the product you are wanting to insert. For example, CAGTTGAATTCGCTCTTCaATG for the
forward oligonucleotide for cloning into the FP site, and GCAAGCTTGCTCTTCtCTT for the reverse
oligonucleotide.
P7.2 PCR amplification of insert
Amplify the fragment using the
general PCR method
and purify using the
general column purification method
.
P7.3 Restriction digestions
Digest ~ 150 ng of both the PCR product and vector with appropriate enzymes.
Set up two restriction digestion reactions (one for vector and one for insert)
8-12
μ
l H20 (for a total of 14
μ
l before addition of enzyme)
1.5
μ
l of 10X restriction buffer
1-4
μ
l of DNA (~150 ng total)
0.75
μ
l Enzyme A
0.75
μ
l Enzyme B
Incubate 1-2 hr at 37°C.
P7.4 Gel puri
fi
cation of DNA fragments
Load the samples on a wide lane 0.8 %
low melt
agarose gel
.
25
Run gel 20-30 minutes.
View the gel under UV (with low iron glass or UV transparent Plexiglas between the gel and UV source to
protect the DNA from damage), and “punch out” each DNA with a 100
μ
l capillary pipette in ~10
μ
l.
P7.5 Ligation
Melt the agarose vector and insert punches at 65°C for 2 minutes.
mix in order
14
μ
l H20
2
μ
l 10X ligase buffer
2
μ
l melted vector
2
μ
l melted insert
1/4
μ
l T4 DNA ligase.
Incubate 0.5 hr. to o.n. at 15°C
P7.6 Transformation into
E. coli
and analysis
Melt the ligation at 65°C and transform using the general
E. coli
transformation method
using 100
μ
l of
competent cells and 1
μ
l of the melted ligation. Colony yield depends greatly on the amount of DNA obtained
from the punch. Our general rule of thumb is that if you can see the DNA, it is enough to get the clone using
this approach. Miniprep 2-4 colonies. Digest DNA with diagnostic enzymes to confirm clone identity.
Sequence if appropriate.
E) Multi-fragment BsaI cloning method
This approach is used either to create more complicated inserts that contain multiple independent
fragments and/or to eliminate
BsaI
or
SapI
sites from templates.
Construction of RMCE targeting plasmids
17