Version 1
Jan 31, 2023
External link: https://doi.org/10.1371/journal.pone.0281625
Protocol Citation: Marc Blanch-Asensio, Sourik Dey, Shrikrishnan Sankaran 2023. In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1o82olr2/v1
MANUSCRIPT CITATION:
Blanch-Asensio M, Dey S, Sankaran S (2023) In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. PLoS ONE 18(2): e0281625. doi: 10.1371/journal.pone.0281625
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: Sep 26, 2022
Last Modified: Jan 31, 2023
PROTOCOL integer ID: 70493
Funders Acknowledgement:
DFG Resarch grant
Grant ID: 455063657
Collaborative Research Centre, SFB 1027
Grant ID: 200049484
Leibniz Science Campus on Living Therapeutic Materials
Grant ID: LifeMat
In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1 V.1 Peer-reviewed method
Peer-reviewed method
- 1Bioprogrammable Materials Group, INM - Leibniz Institute for New Materials, Saarbrücken, Germany.;
- 2Bioprogrammable Materials Group, INM - Leibniz Institute for New Materials, Saarbrücken, Germany
Abstract
Protocol detailing a Gibson-assembly-based direct cloning method for Lactiplatibacillus plantarum WCFS1.
MOLECULAR CLONING
13h
2h
1
VECTOR PCR
Amplify the backbone sequence of interest. Always use a high-fidelity polymerase such as Q5 polymerase. PCR parameters:
- Use 10 ng of DNA as a template.
- Set the initial denaturation time to 1 min.
- Set the denaturation time during the cycles to 10 seconds.
- Extension: 30 sec/kb.
- Set the final extension time to 8 minutes.
- 28-cycle PCR.
- The volume of the reaction = 60 µl.
1h 30m
2
INSERT PCR
Amplify the sequence of interest using overhang primers to add the overhang sequences at the ends of the PCR product. The length of the overhang sequences depends on the commercial kit used for the Gibson Assembly. If the HiFi Assembly Master Mix from New England Biolabs (NEB) is used, an overhang of 20 base pairs is sufficient.
PCR parameters:
- Use 10 ng of DNA as a template.
- Set the initial denaturation time to 1 min.
- Set the denaturation time during the cycles to 10 seconds.
- Extension: 30 sec/kb.
- Set the final extension time to 8 minutes.
- 28-cycle PCR.
- The volume of the reaction = 80 µl.
35m
3
GEL ELECTROPHORESIS
1. Make a 1% agarose gel.
2. Run 5 µl of each PCR on an agarose gel electrophoresis to confirm amplification at the expected size. Run the gel for 30 min at 100 V.
30m
4
DNA PURIFICATION
1. Purify the PCR using the Wizard® SV Gel and PCR Clean-Up System from Promega following the protocol suggested by the manufacturer.
2. Measure the DNA concentration using a NanoDrop Microvolume UV-Vis Spectrophotometer.
40m
5
GIBSON ASSEMBLY
Assemble the vector and the insert using the HiFi Assembly Master Mix from New England Biolabs (NEB).
1. Define the amounts of vector and insert DNA required in this reaction following the protocol suggested by the manufacturer.
2. Mix the linear vector and insert DNA with overlapping DNA fragments with 10 µl of the HiFi DNA Assembly Master Mix.
3. Add Milli-Q water up to 25 µl.
4. Incubate the reaction at 50 °C for 30 minutes.
2h
6
ASSEMBLED PRODUCT PCR
Amplify the assembled product by PCR. Use a high-fidelity PCR such as Q5 polymerase.
1. Design primers that anneal to the insert and cover the whole assembled plasmid. An especially good set of primers is needed for this reaction. Properly check the primers with a primer design tool.
2. Use 5 µl of the HiFi Assembly reaction as a template for amplification. PCR parameters:
- Set the initial denaturation time to 2 min.
- Set the denaturation time during the cycles to 10 seconds.
- Extension: 30 sec/kb.
- Set the final extension time to 8 minutes.
- 23-cycle PCR.
- The volume of the reaction = 120 µl.
35m
7
GEL ELECTROPHORESIS
1. Make a 1% agarose gel.
2. Run 5 µl of each PCR on an agarose gel electrophoresis to confirm amplification at the expected size. Run the gel for 30 min at 100 V.
30m
8
DNA PURIFICATION
1. Purify the PCR using the Wizard® SV Gel and PCR Clean-Up System from Promega following the protocol suggested by the manufacturer.
2. Measure the DNA concentration using a NanoDrop Microvolume UV-Vis Spectrophotometer.
45m
9
QUICK BLUNTING REACTION (PHOSPHORYLATION)
Phosphorylate the 5´ends of the purified PCR products using the Quick Blunting Kit from NEB.
1. Mix 3500 ng of the purified DNA with 2.5 µl of the 10X Quick Blunting buffer and 1 µl of the Enzyme Mix.
2. Add Milli-Q water up to 25 µl.
3. Incubate the reaction first at 25 °C for 30 min.
4. Incubate the reaction at 70 °C for 10 min to inactivate the enzymes.
3h
10
LIGATION
Ligate the phosphorylated products using the T4 Ligase enzyme from NEB.
1. Mix 500 ng of the phosphorylated DNA (3.6 µl of the Quick Blunting reaction) with 1.5 µl of T4 Ligase and 2.5 µl of 10X T4 Ligase Buffer.
2. Add Milli-Q water up to 25 µl.
3. Incubate the reaction first at 25 °C for 2.5 hours.
4. Incubate the reaction at 70 °C for 10 min to inactivate the enzymes.
The number of ligation reactions differs based on the amount of DNA that is desired to be circularized.
35m
11
DNA PURIFICATION
1. Purify the PCR using the Wizard® SV Gel and PCR Clean-Up System from Promega following the protocol suggested by the manufacturer except for the elution step. Perform the elution three times (each time with 9 µl of Mili-Q water) in order to concentrate the ligated product.
2. Measure the DNA concentration using a NanoDrop Microvolume UV-Vis Spectrophotometer.
DNA is ready to be transformed.
LACTIPLANTIBACILLUS PLANTARUM WCFS1 ELECTROCOMPETENT CELLS PREPARATION
18h 15m
5m
12
INOCULATION WILD-TYPE BACTERIA
Inoculate Wild-type L. plantarum WCFS1 from glycerol stock in 5 mL of MRS media.
14h
13
INCUBATION INITIAL CULTURE
Incubate the bacterial culture overnight and at 37 °C with shaking (250 rpm).
5m
14
SECONDARY CULTURE
After approximately 16h, add 1 mL of the culture (OD600 = 2) to 20 mL of MRS media and 5 mL of 1% (w/v) glycine.
4h
15
INCUBATION SECONDARY CULTURE
Incubate this secondary culture for roughly 4 h at 37 °C and 250 rpm until OD600 reached 0.8.
1h
16
WASHINGS
1. Harvest the cells by centrifugation at 4000 rpm (3363 X g) for 12 min at 4°C.
2. Manually discard the supernatant.
3. Wash the pellet twice with 5 mL of ice-cold 10 mM MgCl2. Each time centrifuge cells at 4000 rpm (3363 X g) for 10 min at 4°C.
4. Wash the pellet twice (first with 5 mL and then with 1 mL) with ice-cold Suc/Gly solution (1 M sucrose and 10% (v/v) glycerol mixed in a 1:1 (v/v) ratio). Each time centrifuge cells at 4000 rpm (3363 X g) for 10 min at 4°C.
5. Manually discard the supernatant.
6. Resuspend the bacterial pellet in 450 μL of ice-cold Sac/Gly solution.
5m
17
ALIQUOTING
1. Make aliquotes of 60 uL each.
2. Immediately store at -80 °C the aliquots that are not going to be used.
3. Keep the aliquots that are going to be used for the transformation on ice.
ELECTROPORATION OF PLASMID DNA INTO LACTIPLANTIBACILLUS PLANTARUM WCFS1
2d 3h 20m
15m
18
ELECTROPORATION
1. Mix the plasmid DNA (300 – 1200 ng) with 60 µl of freshly prepared electrocompetent cells.
2. Incubate the mixture on ice for 10 minutes.
3. Transfer the mixture to an ice-cold electroporation cuvette with a 2 mm gap (Bio-Rad Laboratories GmbH).
4. Insert the cuvette in the MicroPulser Electroporator (Bio-Rad Laboratories GmbH, Germany).
5. Apply a single pulse (5 ms) at 1.8 kV.
6. Add immediately 1 mL of room-temperature MRS media after the pulse.
7. Transfer the mixture into a 1.5 mL Eppendorf tube.
3h
19
INCUBATION
Incubate the bacteria at 37 °C and 250 rpm for 3 h to allow the expression of antibiotic resistance genes.
5m
20
PLATING
1. After the incubation, centrifuge the cells at 4000 rpm (3363 X g) for 5 min.
2. Discard the 800 μL of the supernatant.
3. Resuspend the remaining 200 μL by slow pipetting.
4. Plate the resuspended pellet on an MRS Agar plate supplemented with 10 μg/mL of Erythromycin.
2d
21
INCUBATION
Incubate the plate at 37 °C for 48 h for colonies to grow.
SEQUENCE VERIFICATION
17h 30m
2h
22
COLONY PCR FOR SEQUENCE VERIFICATION
1. Label the colonies to be screened on the plate.
2. For each colony, take half of the colony using a sterile pipette tip.
3. Scratch off the tip on a PCR tube.
4. Perform the PCR in this tube using the colony as the template for amplifying the gene of interest. Use primers that amplify the whole gene of interest. PCR parameters:
- Set the initial denaturation time to 10 minutes.
- Set the denaturation time during the cycles to 15 seconds.
- Extension: 30 sec/kb.
- Set the final extension time to 8 minutes.
- 28-cycle PCR
- Volume of the reaction = 100 µl.
Alternatively, the sequence verification PCR can also be done using the bacterial pellet instead of a colony. In this case:
1. Take 1 mL of an overnight culture (in a 1.5 mL Eppendorf tube) and centrifuge it at 4°C for 3 min at 8400 X g.
2. Manually discard the supernatant.
3. Scratched off the residual pellet fraction with a sterile pipette tip
4. Perform the PCR in this tube. Same PCR parameters as before.
35m
23
GEL ELECTROPHORESIS
1. Make a 1% agarose gel.
2. Run 5 µl of each PCR on an agarose gel electrophoresis to confirm amplification at the expected size. Run the gel for 30 min at 100 V.
30m
24
DNA PURIFICATION
1. Purify the PCR using the Wizard® SV Gel and PCR Clean-Up System from Promega following the protocol suggested by the manufacturer.
2. Measure the DNA concentration using a NanoDrop Microvolume UV-Vis Spectrophotometer.
15m
25
SANGER SEQUENCING
1. Send at least 1200 ng of purified DNA for sequencing. Higher amounts of DNA result in a better reading.
2. Request an additional DNA purification step by the sequencing company (Eurofins Genomics). Additional Service: PCR Purification.
5m
26
INOCULATION
Take the remaining half of the colony and inoculate it into 5 mL of MRS media supplemented with 10 μg/mL of erythromycin.
14h
27
INCUBATION
Incubate the culture overnight at 37 °C with shaking (250 rpm).
5m
28
GLYCEROL STOCK
Mix 750 µl of the overnight culture with 250 µl of 60% glycerol. Store the tube at -80 °C.