The Multiple Integration Element (MIE) was derived from a polylinker4 and allows for several approaches for multigene assembly. Single or multiple genes can be inserted into the MIE of any of the ACEMBL vectors by a variety of methods. For this, the vector needs to be linearized, which can be carried out efficiently by PCR reaction with appropriate primers, since the vectors are all small (2-2.6 kb). Alternatively, if more conventional approaches are preferred i.e. in a regular wet lab setting without robotics, the vectors can also be linearized by restriction digestion, and a gene of interest can be pasted in by ligation. The following protocols describe these approaches in detail.
Single gene insertion into the MIE by SLIC
- Primer design
Design primers for the SLIC procedure containing the regions of homology which result in the long sticky ends upon treatment with T4 DNA polymerase in the absence of dNTPs3:
Primers for the insert contain a DNA sequence corresponding to this region of homology (adaptor sequence), followed by a sequence which specifically anneals to the insert to be amplified. Useful adaptor sequences for SLIC can be taken directly from the ACEMBL Manual deposited at the EMBL Grenoble homepage: "http://www.embl.fr/research/services/berger/ACEMBL.pdf":http://www.embl.fr/research/services/berger/ACEMBL.pdf.
In case the gene of interest is amplified from a vector already containing expression elements (e.g. the pET vector series), the “insert specific sequence” can be located upstream of a ribosome binding site (rbs). Otherwise, the forward primer needs to be designed such that a ribosome binding site is also provided in the final construct.
Primers for PCR linearization of the vector backbone are simply complementary to the two adaptor sequences present in the primer pair chosen for insert amplification.
- PCR amplification of insert and vector
Prepare PCR reactions in 100 µl volume for DNA insert to be cloned and the vector backbone to be linearized:
ddH2O: 75 µl
5x Phusion HF Reaction buffer: 20 µl
dNTPs (10 mM stock): 2 µl
Template DNA (100 ng/µl): 1 µl
5' SLIC primer (100 µM stock): 1 µl
3' SLIC primer (100 µM stock): 1 µl
Phusion polymerase (2 U/µl): 0.5 µl
Carry out PCR reactions with a standard PCR program (unless very long DNAs are amplified, then double the extension time or refer to the corresponding instruction of the polymerase to be used):
1 x 98 ºC for 2 min
30 x [98 ºC for 20 s → 50 ºC for 30 s → 72 ºC for 3 min]
Hold at 10 ºC
Analysis of the PCR reactions by agarose gel electrophoresis and ethidium bromide staining is recommended.
- DpnI treatment of PCR products (optional)
Supply PCR reactions with 1 µl DpnI enzyme which cleaves parental plasmids (methylated). For insert PCR reactions, DpnI treatment is not required if the resistance marker of the template plasmid differs from the destination vector.
Carry out reactions as follows:
Incubation: 37 ºC for 1-4 h
Inactivation: 80 ºC for 20 min
- Purification of PCR products
Note: PCR products must be cleaned of residual dNTPs. Otherwise, the T4 DNA polymerase reaction (step 5) is compromised.
Product purification is best performed by using commercial kits. It is recommended to perform elution in the minimal possible volume indicated by the manufacturer.
- T4 DNA polymerase exonuclease treatment
Prepare identical reactions in a 20 µl volume for the insert and the corresponding vector it should be cloned into (both eluted in step 4):
10x T4 DNA polymerase buffer: 2 µl
100 mM DTT: 1 µl
2 M Urea: 2 µl
DNA eluate from Step 3 (vector or insert): 14 µl
T4 DNA polymerase: 1 µl
Carry out reactions as follows:
Incubation: 23 ºC for 20 min
Arrest: Addition of 1 µl 500 mM EDTA
Inactivation: 75 ºC for 20 min
- Mixing and Annealing
Mix T4 DNA polymerase treated insert and vector (step 5), followed by an (optional) annealing step which was found to enhance efficiency:
T4 DNA pol treated insert: 10 µl
T4 DNA pol treated vector: 10 µl
Annealing: 65 ºC for 10 min
Cooling: Slowly to RT (at least 2h)
- Transformation
Transform mixture from step 6 into competent cells following standard transformation procedures.
Transform reactions for pACE and pACE2 derivatives into standard E. coli cells for cloning (such as MACH1, TOP10, DH5α, HB101). After recovery (2-4 h) plate the transformed reactions on agar containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Transform reactions for Donor derivatives into E. coli cells expressing the pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plate the transformed reactions on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml, pDK) or spectinomycin (50 µg/ml, pDS).
It is recommended to plate the transformed reaction on two agar plates in dilution series, so that one can always easily pick single colonies after the overnight incubation.
- Plasmid analysis
Grow culture for plasmid isolation (small-scale) in media containing the corresponding antibiotic. The isolated plasmids should then be analyzed by sequencing and (optional) restriction mapping using appropriate restriction enzymes.
Polycistron assembly in MIE by SLIC
The multiple integration element (MIE) can also be used to integrate genes of interest by using multi-fragment SLIC recombination in order to assemble polycistrons. Genes preceded by ribosome binding sites (rbs) can be assembled in this way under the control of one promoter.
- Primer design
The multiple integration element (MIE) is composed of tried-and-tested primer sequences. These constitute the “adaptor sequences” that can be used for inserting single genes or multigene constructs. Recommended adaptor sequences for SLIC can be taken directly from the ACEMBL manual: "http://www.embl.fr/research/services/berger/ACEMBL.pdf":http://www.embl.fr/research/services/berger/ACEMBL.pdf.
Adaptor sequences form the 5’ segments of the primers used to amplify DNA fragments to be inserted into the MIE. Insert specific sequences are added at 3’, and a DNA sequence encoding for a ribosome binding sites can be inserted optionally if not already present on the PCR template.
- PCR amplification of inserts and vector
Prepare identical PCR reactions in 100 µl volume for all inserts to be cloned and the vector backbone to be linearized:
ddH2O: 75 µl
5x Phusion HF Reaction buffer: 20 µl
dNTPs (10 mM stock): 2 µl
Template DNA (100 ng/µl): 1 µl
5' SLIC primer (100 µM stock): 1 µl
3' SLIC primer (100 µM stock): 1 µl
Phusion polymerase (2 U/µl): 0.5 µl
Carry out PCR reactions with a standard PCR program (unless very long DNAs are amplified, then double extension time or refer to the corresponding instruction of the polymerase to be used):
1 x 98 ºC for 2 min
30 x [98 ºC for 20 s → 50 ºC for 30 s → 72 ºC for 3 min]
Hold at 10 ºC
Analysis of the PCR reactions by agarose gel electrophoresis and ethidium bromide staining is recommended.
- DpnI treatment of PCR products (optional)
Supply PCR reactions with 1 µl DpnI enzyme which cleaves parental plasmids (methylated). For insert PCR reactions, DpnI treatment is not required if the resistance marker of the template plasmids differs from the destination vector.
Carry out reactions as follows:
Incubation: 37 ºC for 1-4h
Inactivation: 80 ºC for 20 min
- Purification of PCR products
Note: PCR products must be cleaned of residual dNTPs. Otherwise, the T4 DNA polymerase reaction (step 5) is compromised.
Product purification is best performed by using commercial kits. It is recommended to perform elution in the minimal possible volume indicated by the manufacturer.
- T4 DNA polymerase exonuclease treatment
Prepare identical reactions in 20 µl volume for each insert and the corresponding vector they should be cloned into (both eluted in step 4):
10x T4 DNA polymerase buffer: 2 µl
100 mM DTT: 1 µl
2M Urea: 2 µl
DNA eluate from Step 3 (vector or insert): 14 µl
T4 DNA polymerase: 1 µl
Carry out reactions as follows:
Incubation: 23 ºC for 20 min
Arrest: Addition of 1 µl 500 mM EDTA
Inactivation: 75 ºC for 20 min
- Mixing and Annealing
Mix T4 DNA polymerase treated inserts and vector (step 5), followed by an (optional) annealing step which was found to enhance efficiency1:
T4 DNA pol. treated insert 1: 5 µl
T4 DNA pol. treated insert 2: 5 µl
T4 DNA pol. treated insert 3: 5 µl
T4 DNA pol. treated vector: 5 µl
Annealing: 65 ºC for 10 min
Cooling: Slowly (switch off heat block) to RT
- Transformation
Transform mixture from step 6 into competent cells following standard transformation procedures.
Transform reactions for pACE and pACE2 derivatives into standard E. coli cells for cloning (such as MACH1, TOP10, DH5α, HB101). After recovery, plate the transformed reactions on agar containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Transform reactions for Donor derivatives into E. coli cells expressing the pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plate the transformed reactions on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml, pDK) or spectinomycin (50 µg/ml, pDS).
It is recommended to plate the transformed reaction on two agar plates in dilution series, so that one can always easily pick single colonies after the overnight incubation.
- Plasmid analysis
Grow culture for plasmid isolation in media containing the corresponding antibiotic. The isolated plasmids should then be analyzed by sequencing and (optional) restriction mapping using appropriate restriction enzymes.
Gene insertion by restriction/ligation
- Primer design
For conventional cloning, if the gene of interest is to be PCR amplified, design PCR primers containing chosen restriction sites, preceded by appropriate overhangs for efficient restriction digestion (c.f. New England Biolabs catalogue). This region is followed by ≥ 20 nucleotides overlapping with the gene of interest that is to be inserted.
MIEs are identical in all the ACEMBL vectors. They contain a ribosome binding site preceding the NdeI site. Therefore, for single gene insertions, a ribosome binding site (rbs) does not need to be included in the forward primer.
In case multigene insertions are planned, primers need to be designed such that a rbs is at the beginning of the gene and a stop codon at its end. Therefore, in particular for polycistron cloning by restriction/ligation, it is recommended to construct templates by custom gene synthesis. In this process, the restriction sites present in the MIE can be eliminated from the encoding DNAs.
- Insert preparation
i) PCR of insert(s):
Prepare identical PCR reactions in 100 µl volume for each gene of interest to be inserted into the MIE:
ddH2O: 75 µl
5x Phusion HF Reaction buffer: 20 µl
dNTPs (10 mM stock): 2 µl
Template DNA (100 ng/µl): 1µl
5' primer (100 µM stock): 1 µl
3' primer (100 µM stock): 1 µl
Phusion polymerase (2 U/µl): 0.5 µl
Carry out PCR reactions with a standard PCR program (unless very long DNAs are amplified, then double the extension time or refer to the corresponding instruction of the polymerase used):
1 x 98 ºC for 2 min
30 x [98 ºC for 20 s → 50 ºC for 30 s → 72 ºC for 3 min]
Hold at 10 ºC
Analysis of the PCR reactions by agarose gel electrophoresis and ethidium bromide staining is recommended.
Purification of PCR products is best performed by using commercial kits. It is recommended to perform elution in the minimal possible volume indicated by the manufacturer.
ii) Restriction digestion of insert(s):
Carry out restriction reactions in 40 µl reaction volume, by using the specific restriction enzymes as specified by manufacturer’s recommendations.
PCR Kit eluate (≥ 1 µg): 30 µl
10x Restriction enzyme buffer: 4 µl
10 mM BSA: 2 µl
Restriction enzyme for 5’: 2 µl
Restriction enzyme for 3’: 2 µl (in case of double digestion, otherwise ddH2O)
Perform restriction digestion in a single reaction with both enzymes (double digestion) or sequentially (two single digestion reactions) if the reaction conditions required are incompatible.
iii) Gel extraction of insert(s):
Purify processed inserts by agarose gel extraction using commercial kits. It is recommended to elute the extracted DNA in the minimal volume defined by the manufacturer.
- Vector preparation
i) Restriction digestion of ACEMBL plasmid(s):
Carry out restriction reactions in 40 µl reaction volume, using specific restriction enzymes as specified by manufacturer’s recommendations (c.f. New England Biolabs catalogue and others).
ACEMBL plasmid (≥ 0.5 µg) in ddH2O: 30 µl
10x Restriction enzyme buffer: 4 µl
10 mM BSA: 2 µl
Restriction enzyme for 5’: 2 µl
Restriction enzyme for 3’: 2 µl (in case of double digestion, otherwise ddH2O)
Perform restriction digestion in a single reaction with both enzymes (double digestion) or sequentially (two single digestion reactions) if the reaction conditions required are incompatible.
Analysis of the restriction digestion of ACEMBL vectors by agarose gel electrophoresis and ethidium bromide staining is recommended before gel extraction (ii).
ii) Gel extraction of linearized vector(s):
Purify processed vectors by agarose gel extraction using commercial kits. It is recommended to elute the extracted DNA in the minimal volume defined by the manufacturer.
- Ligation
It is recommended to analyze the intensity and integrity of vectors and inserts from gel extraction by agarose gel electrophoresis and ethidium bromide staining. Normally the ratio between vector and insert is ranged from 1:3 to 1:6.
Carry out ligation reactions in 20 µl reaction volume according to the recommendations of the supplier of T4 DNA ligase:
ACEMBL plasmid (gel extracted, step 3): 8 µl
Insert (gel extracted, step 2): 10 µl
10x T4 DNA Ligase buffer: 2 µl
T4 DNA Ligase: 0.5 µl
Perform ligation reactions at 25 ºC (sticky end) for 1h or at 16 ºC (blunt end) overnight.
- Transformation
Transform ligation mixtures (step 4) into E. coli competent cells following standard transformation procedures.
Transform reactions for pACE and pACE2 derivatives into standard E. coli cells for cloning (such as TOP10, DH5α, HB101). After recovery, plate the transformed reactions on agar containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Transform reactions for Donor derivatives into E. coli cells expressing the pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plate the transformed reactions on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml, pDK) or spectinomycin (50 µg/ml, pDS).
We recommend plating the transformed reaction on agar plates in a dilution series, to ensure optimal colony separation.
- Plasmid analysis
Culture plasmids and select correct clones based on specific restriction digestion and DNA sequencing of the inserts.
Multiplication by using the HE and BstXI sites
The presence of a homing endonuclease (HE) cutting site (PI-SceI or I-CeuI) together with a BstXI site makes it feasible to iteratively insert further gene(s) of interest, which are already cloned into the MIE of an ACEMBL vector, into the expression cassette. The insert is being released by restriction digestion with both HE and BstXI, whereas the vector is being linearized by restriction digestion with HE.
- Insert preparation
i) Restriction digestion of insert(s)
Carry out restriction reactions in 40 µl reaction volume by using homing endonucleases PI-SceI (Donors) or I-CeuI (Acceptors) as recommended by the supplier (c.f. New England Biolabs catalogue and others).
ACEMBL plasmid (≥ 0.5 µg) in ddH2O: 32 µl
10x Restriction enzyme buffer: 4 µl
10 mM BSA: 2 µl
PI-SceI (Donors) or I-CeuI (Acceptors): 2 µl
Purify reactions using commercial kits, or acidic ethanol precipitation and perform the second restriction digestion by BstXI according to the recommendations of the supplier.
HE digested DNA in ddH2O: 32 µl
10x Restriction enzyme buffer: 4 µl
10 mM BSA: 2 µl
BstXI: 2 µl
ii) Gel extraction of insert(s):
Purify processed insert(s) by agarose gel extraction using commercial kits. It is recommended to elute the extracted DNA in the minimal volume defined by the manufacturer.
- Vector preparation
i) Restriction digestion of vector(s)
Carry out restriction reactions in 40 µl reaction volume by using homing endonucleases PI-SceI (Donors) or I-CeuI (Acceptors) as recommended by the supplier (c.f. New England Biolabs catalogue and others).
ACEMBL plasmid ( ≥ 0.5 µg) in ddH2O: 33 µl
10x Restriction enzyme buffer: 4 µl
10 mM BSA: 2 µl
PI-SceI (Donors) or I-CeuI (Acceptors): 1 µl
Analysis of restriction digestion of ACEMBL vectors by agarose gel electrophoresis and ethidium bromide staining before phosphatase treatment is recommended.
Purify reactions using commercial kits, or acidic ethanol precipitation. Next, treat the purified reactions with intestinal alkaline phosphatase according to the recommendations of the supplier.
HE digested DNA in ddH2O: 17 µl
10x Alkaline phosphatase buffer: 2 µl
Alkaline phosphatase: 1 µl
ii) Gel extraction of vector(s):
Purify processed vector(s) by agarose gel extraction using commercial kits. It is recommended to elute the extracted DNA in the minimal volume defined by the manufacturer.
- Ligation
It is recommended to analyze the intensity and integrity of vectors and inserts from gel extraction by agarose gel electrophoresis and ethidium bromide staining. Normally the ratio between vector and insert is ranged from 1:3 to 1:6.
Carry out ligation reactions in 20 µl reaction volume:
HE/Phosphatase treated vector (gel extracted): 4 µl
HE/BstXI treated insert (gel extracted): 14 µl
10x T4 DNA Ligase buffer: 2 µl
T4 DNA Ligase: 0.5 µl
Perform ligation reactions at 25 ºC for 1h or at 16 ºC overnight.
- Transformation
Transform ligation mixtures from step 3 into E. coli competent cells following standard transformation procedures.
Transform reactions for pACE and pACE2 derivatives into standard E. coli cells for cloning (such as TOP10, DH5α, HB101). After recovery, plate the transformed reactions on agar containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Transform reactions for Donor derivatives into E. coli cells expressing the pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plate the transformed reactions on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml, pDK) or spectinomycin (50 µg/ml, pDS).
We recommend plating the transformed reaction on two agar plates in dilution series, to ensure optimal colony separation.
- Plasmid analysis
Culture plasmids and select correct clones based on specific restriction digestion and DNA sequencing of the inserts.
Note: One can likewise perform the integration by sequence and ligation independent cloning (SLIC). It is recommended to carry out linearization of the vector by digestion with HE, if heterologous genes are already present, to avoid PCR amplification over encoding regions. The fragment to be inserted is generated by PCR amplification resulting in a PCR fragment containing a 20-25 base pair stretch at its 5’ end that is identical to the corresponding DNA sequence present at the HE site counted from the site of cleavage towards 5’ (site of cleavage is position -4). At the 3’ end of the PCR fragment, the homology region is 20-25 base pairs counted from the site of cleavage towards 3’.
Cre-LoxP fusion of Acceptors and Donors
Cre recombinase is a member of the integrase family catalyzing the recombination of a 34 bp LoxP site in the absence of accessory protein or auxiliary DNA sequence. The LoxP site itself is comprised of two 13 bp recombinase-binding elements arranged as inverted repeats flanking an 8 bp central region where cleavage and ligation reaction occur. As all ACEMBL plasmids contain a single LoxP site, they can be fused in a Cre-dependent reaction. This is possible not only for 2 plasmids (Acceptor-Donor fusion), but also for the fusion of several (3-4) plasmids in a single reaction.
The fact that Donors contain a conditional origin of replication that depends on a pir+ background allows for selection of desired fusion products out of such a reaction. Being transformed into pir- strains (MACH1, TOP10, DH5α, HB101 or other common laboratory cloning strains), Donor vectors will act as suicide vectors when plated out on agar containing the antibiotic corresponding to the Donor encoded resistance marker, unless fused with an Acceptor. By properly combining antibiotics in the agar, all desired Acceptor-Donor fusions can be selected.
- For a 20 µl Cre reaction, mix 1-2 µg of each educt in approximately equal amounts. Add ddH2O to adjust the total volume to 16-17 µl, then add 2 µl 10x Cre buffer and 1-2 µl Cre recombinase.
CRITICAL STEP
Incubate Cre reaction at 37 °C (or 30 °C) for 1 hour.
Optional: load 2-5 µl of Cre reaction on an analytical agarose gel for examination.
Note: Heat inactivation at 70 °C for 10 minutes before the gel loading is strongly recommended.
- For chemical transformation, mix 10-15 µl Cre reaction with 200 µl chemical competent cells. Incubate the mixture on ice for 15-30 minutes. Then perform heat shock at 42 °C for 45-60 s.
Note: Up to 20 µl Cre reaction (max. 10% of the total volume of chemical competent cell suspension) can be directly transformed into 200 µl chemical competent cells. For electro-transformation, one could mix up to 2 µl Cre reaction with 100 µl electrocompetent cells and perform the transformation by using an electroporator (e.g. BIORAD E. coli Pulser) at 1.8-2.0 kV.
Note: Larger volumes of Cre reaction must be desalted by ethanol precipitation or a PCR purification column before electrotransformation. The desalted Cre reaction mix should not exceed 10% of the volume of the electrocompetent cell suspension.
The cell/DNA mixture could be immediately used for electrotransformation without prolonged incubation on ice.
Add up to 400 µl of LB media (or SOC media) per 100 µl of cell/DNA suspension immediately after the transformation (heat shock or electroporation).
Incubate the suspension in a 37 °C shaking incubator overnight or for at least 4 hours (recovery period).
Note: For recovering multifusion plasmid containing more than 2 resistance markers, it is strongly recommended to incubate the suspension at 37 °C overnight.
- Plate out the recovered cell suspension on agar containing the desired combination of antibiotics. Incubate at 37 °C overnight.
TROUBLESHOOTING
- Clones from colonies present after overnight incubation can be verified by restriction digestion at this stage (refer to steps 12-16).
Note: Verification is recommended especially in the case that only one multifusion plasmid is desired.
For further selection by single antibiotic challenges on a 96 well microtiter plate, continue to step 9.
Note: Several to many different multifusion plasmid combinations can be processed and selected on one 96 well microtiter plate in parallel.
For 96 well antibiotic tests, inoculate four colonies from each agar plate with different antibiotic combination into ~500 µl LB media without antibiotics. Incubate the cell cultures in a 37 °C shaking incubator for 1-2 hours.
During the incubation of colonies, fill a 96 well microtiter plate with 150 µl antibiotic-containing LB media. We added coloured dye (positional marker) in selected wells as positional markers (Fig. 2).
Note: A typical arrangement of the solutions, which is used for parallel selection of multifusion plasmids, is shown in Figure 2 as well as the ACEMBL Manual: "http://www.embl.fr/research/services/berger/ACEMBL.pdf":http://www.embl.fr/research/services/berger/ACEMBL.pdf. The concept behind the 96 well plate experiment is that every cell suspension from single colonies needs to be challenged by all four single antibiotics for unambiguous interpretation.
- Add 1 µl aliquots of pre-incubated cell culture (Step 9) to the corresponding wells. Then incubate the inoculated 96 well microtiter plate in a 37 °C shaking incubator overnight at 180-200 rpm.
Recommended: Use parafilm to wrap the plate to avoid drying out.
The remainder of the pre-incubated cell cultures could be kept at 4 °C for further inoculation if necessary.
Select transformants containing desired multifusion plasmids based on antibiotic resistance, according to the combination of dense (positive) and clear (no growth) cell microcultures from each colony. Inoculate 10-20 µl cell culture into 10 ml LB media with corresponding antibiotics. Incubate in a 37 °C shaking incubator overnight.
Centrifuge the overnight cell cultures at 4000 g for 5-10 minutes. Purify plasmid from the resulting cell pellets. It is recommended to utilize commercial kits.
Determine the concentration of purified plasmid solutions by using UV absorption spectroscopy (e.g. by using a NanoDropTM 1000 machine).
Digest 0.5-1 µg of the purified plasmid solution in a 20 µl restriction digestion with appropriate endonuclease(s). Incubate under recommended reaction condition for ~2 hours.
Use 5-10 µl of the digestion for analytical agarose (0.8-1.2 %) gel electrophoresis. Verify plasmid integrity by comparing the experimental restriction pattern to a restriction pattern predicted in silico (e.g. by using program VectorNTI from Invitrogen or similar programs).
Deconstruction of fusion vectors by Cre recombinase
It is advantageous to release all or part of the educts composing a particular multifusion plasmid, for further modification and diversification.
Incubate ==== 1 µg multifusion plasmid with 2 µl 10x Cre buffer and 1-2 µl Cre recombinase. Add ddH2~O to adjust the total reaction volume to 20 µl.
Incubate this Cre deconstruction reaction mixture at 30°C (1-4 h).
Optional: load 2-5 µl of the reaction on an analytical agarose gel for examination.
Note: Heat inactivation at 70 °C for 10 minutes before the gel loading is strongly recommended.
- For chemical transformation, mix 10-15 µl De-Cre reaction with 200 µl chemical competent cells. Incubate the mixture on ice for 15-30 minutes. Then perform heat shock at 42 °C for 45-60 seconds.
Note: Up to 20 µl De-Cre reaction (10% of total volume of transformation reaction) can be directly transformed into 200 µl chemical competent cells. For electrotransformation, up to 2 µl De-Cre reaction could be directly mixed with 100 µl electrocompetent cells, and transformed by using an electroporator (e.g. BIORAD E. coli Pulser) at 1.8-2.0 kV.
Note: Larger volume of De-Cre reaction must be desalted by ethanol precipitation or PCR purification column before electrotransformation. The desalted De-Cre reaction mix should not exceed 10% of the volume of the electrocompetent cell suspension.
The cell/DNA mixture could be immediately used for electro-transformation without prior incubation on ice.
Add up to 400 µl of LB media (or SOC media) per 100 µl of cell/DNA suspension immediately after the transformation (heat shock or electroporation).
Incubate the suspension in a 37 °C shaking incubator (recovery).
Note: For recovery of partially deconstructed double/triple fusions, incubate the suspension in a 37 °C shaking incubator overnight or for at least 4 hours.
For recovery of individual educts such as single ACEMBL vectors from pACKS plasmid, incubate the suspension in a 37 °C shaking incubator (1-2 h).
- Plate out the recovered cell suspension on agar containing the desired (combination of) antibiotic(s). Incubate at 37 °C overnight.
TROUBLESHOOTING
- Colonies after overnight incubation might be verified directly by restriction digestion at this stage (refer to steps 12-16).
Note: Especially recommended in the case that only one single educt or partially deconstructed multifusion plasmid is desired.
For further selection by single antibiotic challenge on a 96 well microtiter plate, continue with step 9.
Note: Several different single educts/partially deconstructed multifusion plasmids can be processed and selected on one 96 well microtiter plate in parallel.
For 96 well microtiter plate analysis inoculate four colonies each from agar plates containing a defined set of antibiotics into ~500 µl LB media without antibiotics. Incubate the cell cultures in a 37 °C shaking incubator (1-2 h).
During the incubation of colonies, fill a 96 well microtiter plate with 150 µl antibiotic-containing LB media or coloured dye (positional marker) in the corresponding wells (Fig. 2).
Note: Compare Figure 2 as well as the ACEMBL Manual: "http://www.embl.fr/research/services/berger/ACEMBL.pdf":http://www.embl.fr/research/services/berger/ACEMBL.pdf for the arrangement of the solutions in the wells, which are used for parallel selection of single educts or partially deconstructed multifusion plasmids. The concept is that every cell suspension from a single colony needs to be challenged by all four antibiotics separately for unambiguous interpretation.
- Add 1 µl aliquots from the pre-incubated cell cultures (step 9) into the corresponding wells. Then incubate the 96 well microtiter plate in a 37 °C shaking incubator overnight at 180-200 rpm.
Recommended: Use parafilm to wrap the plate to prevent dehydration.
The remainder of the pre-incubated cell cultures can be kept in 4°C fridge for further inoculation if necessary.
Select transformants containing desired single educts or partially deconstructed multifusion plasmids according to the combination of dense (growth) and clear (no growth) cell cultures from each colony. Inoculate 10-20 µl cell cultures into 10 ml LB media with corresponding antibiotic(s). Incubate in a 37 °C shaking incubator overnight.
Centrifuge the overnight cell cultures at 4000 g for 5-10 minutes. Purify plasmid from cell pellets.
Determine the concentration of purified plasmid solutions by using UV absorption spectroscopy (e.g. NanoDropTM 1000).
Digest 0.5-1 µg of the purified plasmid solution in a 20 µl restriction digestion (with 5-10 unit endonuclease). Incubate under recommended reaction condition for ~2 hours.
Use 5-10 µl of the digestion for analytical agarose gel (0.8-1.2 %) electrophoresis. Verify the plasmid integrity by comparing the actual restriction pattern to predicted restriction pattern in silico (e.g. by using VectorNTI, Invitrogen, or any other similar program).
Optional: Possibly, a deconstruction reaction is not complete but yields partially deconstructed fusions which still retain entities to be eliminated. In this case, we recommend to pick these partially deconstructed fusions containing and perform a second round of Cre deconstruction reaction (repeat steps 1-8) by using this construct as starting material.
Note: In our hands, two sequential deconstruction reactions were always sufficient to recover all individual modules.