Sep 21, 2021
Cloning into pSL2680 CRISPR Plasmid - iGEM IISER Pune 2021
- 1Indian Institute of Science Education and Research, Pune
- iGEM IISER Pune India 2021
Protocol Citation: misaal.bedi 2021. Cloning into pSL2680 CRISPR Plasmid - iGEM IISER Pune 2021. protocols.io https://dx.doi.org/10.17504/protocols.io.bw9kph4w
Manuscript citation:
Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Ungerer J, Pakrasi HB. Sci Rep. 2016 Dec 21;6:39681. doi: 10.1038/srep39681. 10.1038/srep39681 PubMed 28000776
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: In development
We are still developing and optimizing this protocol
Created: August 10, 2021
Last Modified: September 21, 2021
Protocol Integer ID: 52236
Abstract
This protocol can be used to clone the guide RNA and Homologous Repair Template into the pSL2680 plasmid which was a gift from Himadri Pakrasi (Addgene plasmid # 85581 ; http://n2t.net/addgene:85581 ; RRID:Addgene_85581).
Materials
pSL2680 was a gift from Himadri Pakrasi (Addgene plasmid # 85581 ; http://n2t.net/addgene:85581 ; RRID:Addgene_85581)
Restriction enzymes: AarI, KpnI + their buffers
Antibiotics: Kanamycin
LB media
Distilled Water
Plasmid Miniprep Kit
Gel Purification Kit
Gel electrophoresis set up
Ligase buffer
PNK Enzyme
NEB Gibson Assembly Kit
Cloning the gRNA
Cloning the gRNA
The gRNA is to be cloned before the cassette into the plasmid.
Day 1
Day 1
Take 30 mL of LB media with 0.05 µg/µL Kanamycin and scratch off some solid culture from the vial and add to the media in a flask. Grow this Overnight at 37 °C
Day 2
Day 2
Perform miniprep on the entire 30 mL culture (since the pSL2680 plasmid is of a low copy number). Spin it down and resuspend it in 500 µL resuspension buffer in the fridge. Divide it into two sets and follow the rest of the miniprep protocol. In the end, elute from the column with 50 µL of distilled water.
Digest the 50 µL of miniprep with AarI using the following reaction mix:
AarI buffer - 10 µL
plasmid prep - 50 µL
50X AarI oligo - 2 µL
Aarl enzyme - 4 µL
Distilled water - 34 µL
Incubate at 37 °C for 4 hours.
Gel extract the digest. Run the entire digest in a double or triple lane on a 0.7-1% agarose gel. Excise the largest band and place it in a 2 mL Eppendorf tube. Melt at 55 °C (Tip: Inverting the tube every five minutes helps in faster melting)
Perform gel purification using a gel purification kit and follow its protocol.
In the end, elute with 20 µL of water (ensure that a small amount of water is placed in the center of the membrane).
Day 3
Day 3
Anneal the gRNA oligos with the following reaction mixture:
100 micromolar (µM) stock of the gRNA left primer - 10 µL
100 micromolar (µM) stock of the gRNA right primer - 10 µL
Ligase buffer - 5 µL
Distilled water - 25 µL
PNK enzyme - 1 µL
Use the following thermocycler program:
Heat to 95 °C for 5 minutes, and then ramp to 4 °C at 0.1 °C /second.
Dilute the annealed oligos 1:50. Not a lot is required so 2 µL with 98 µL of water can be used.
Ligate the oligos into pSL2680 which was digested the previous day with the following reaction mix:
pSL2680 gel extract - 8 µL
Annealed oligos that were dilute 1:50 - 0.5 µL
Ligase buffer - 1 µL
Ligase - 0.5 µL
Incubate Overnight at 16 °C
Day 4
Day 4
Transform the entire 10 µL ligation reaction into the E. coli XL1 - blue or Top10 strains as follows:
Thaw cells On ice for 10 minutes.
Add 10 µL ligation reaction to cells and stir. Don't pipette up and down.
Incubate On ice for another 10 minutes.
Heat shock for 45 seconds at 42 °C .
Place On ice for 2 minutes.
Add 500 µL of liquid LB media.
Incubate at 37 °C for 1 hour.
Centrifuge the reaction mixture after incubation and remove the supernatant. Add the pellet to 50 µL of the media.
Plate transformants on LB media with 0.05 µg/µL Kanamycin and add IPTG+X-gal.
Spread 40 µL of 20 millimolar (mM) IPTG and 20 mg/mL X-gal on a plate with LB media with 0.05 µg/µL Kanamycin and let it soak in for at least an hour.
Prepare the plates before starting the transformation.
Day 5
Day 5
Pick 8 white colonies from the transformants and patch them on LB media with 0.05 µg/µL Kanamycin.
Start 10 mL cultures of 2 colonies off the plate in LB media with 0.05 µg/µL Kanamycin.
Day 6
Day 6
Isolate the plasmid from the 2 cultures incubated the previous day. Determine the concentration and send for sequencing. Once the correct sequence is confirmed, prepare stocks.
Cloning of cassette
Cloning of cassette
After the gRNA has been cloned into the plasmid, we can begin cloning the cassette to make modifications in the S. elongatus UTEX 2973 genome.
Day 1
Day 1
Synthesize the repair template using high-fidelity PCR.
Gel extract the PCR reactions and purify them, and elute in 25 µL of distilled water.
Digest the plasmid carrying the gRNA using Kpnl and make sure to dephosphorylate it, to prevent recircularization of the backbone.
Use the following reaction mixture:
Buffer - 15 µL
Plasmid Prep - 75 µL
Kpnl restriction enzyme - 3 µL
FastAP (dephosphorylates vector) -
Distilled Water - 53 µL
Incubate for 4 hours at 37 °C
Gel extract the digest and elute in 25 µL of distilled water.
Day 2
Day 2
Concentrate the gel extractions and perform Gibson Assembly and concentrate the DNA.
Allow samples to dry for 2.5-3 hours.
For Gibson Assembly, exactly 5 µL of correctly mixed and highly concentrated DNA is required. Use 200 ng
of the vector and a 2X molar ratio of each fragment to be assembled by Gibson Assembly.
Note: Use molar ratios of the fragments, not weight.
The vector and PCR fragments should be mixed using the
Prepare the mixture in a PCR tube with a total volume of 5 µL .
Add 15 µL of the Gibson master mix to the 5 µL of DNA.
Mix them together and PCR at 50 °C for an hour and then hold at 4 °C
Day 3
Day 3
Transform at least 10 µL of the Gibson reaction in XL1-blue (or Top10) using the same protocol as described in step 5 of Day 4 in the 'Cloning the gRNA' section of this protocol.
Plate on LB media with 0.05 µg/µL Kanamycin
Day 4
Day 4
Check the transformed colonies by PCR.
Run the reactions on a gel and select two positive colonies.
Start with 10 mL of each colony in LB media with 0.05 µg/µL Kanamycin for plasmid isolation.
Day 5
Day 5
Isolate plasmids from the two colonies isolated the previous day and elute in 50 µL .
Sequence the insert in the plasmid. If the sequencing is correct, this protocol has been successfully completed and all that remains is to transform the plasmid into cyanobacteria.
Note: Don't forget to freeze the recombinant E.coli in permanent stock.