Cloning into pSL2680 CRISPR Plasmid - iGEM IISER Pune 2021

The gRNA is to be cloned before the cassette into the plasmid.

Take 30mL of LB media with 0.05µg/µL Kanamycin and scratch off some solid culture from the vial and add to the media in a flask. Grow this at 37°C

Perform miniprep on the entire 30mL culture (since the pSL2680 plasmid is of a low copy number). Spin it down and resuspend it in 500µL 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 50µL of distilled water.

Digest the 50µL of miniprep with AarI using the following reaction mix:

AarI buffer - 10µL

plasmid prep - 50µL

50X AarI oligo - 2µL

Aarl enzyme - 4µL

Distilled water - 34µL

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 2mL 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 20µL of water (ensure that a small amount of water is placed in the center of the membrane).

Anneal the gRNA oligos with the following reaction mixture:

100micromolar (µM) stock of the gRNA left primer - 10µL

100micromolar (µM) stock of the gRNA right primer - 10µL

Ligase buffer - 5µL

Distilled water - 25µL

PNK enzyme - 1µL

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 2µL with 98µL of water can be used.

Ligate the oligos into pSL2680 which was digested the previous day with the following reaction mix:

pSL2680 gel extract - 8µL

Annealed oligos that were dilute 1:50 - 0.5µL

Ligase buffer - 1µL

Ligase - 0.5µL

Incubate at 16°C

Transform the entire 10µL ligation reaction into the E. coli XL1 - blue or Top10 strains as follows:

Thaw cells On ice for 10 minutes.

Add 10µL 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 500µL 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 50µL of the media.

Plate transformants on LB media with 0.05µg/µL Kanamycin and add IPTG+X-gal.

Spread 40µL of 20millimolar (mM) IPTG and 20mg/mL X-gal on a plate with LB media with 0.05µg/µL Kanamycin and let it soak in for at least an hour.

Prepare the plates before starting the transformation.

Pick 8 white colonies from the transformants and patch them on LB media with 0.05µg/µL Kanamycin.

Start 10mL cultures of 2 colonies off the plate in LB media with 0.05µg/µL Kanamycin.

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.

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.

Synthesize the repair template using high-fidelity PCR.

Gel extract the PCR reactions and purify them, and elute in 25µL 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 - 15µL

Plasmid Prep - 75µL

Kpnl restriction enzyme - 3µL

FastAP (dephosphorylates vector) -

Distilled Water - 53µL

Incubate for 4 hours at 37°C

Gel extract the digest and elute in 25µL of distilled water.

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 5µL of correctly mixed and highly concentrated DNA is required. Use 200ng

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 5µL .

Add 15µL of the Gibson master mix to the 5µL of DNA.

Mix them together and PCR at 50°C for an hour and then hold at 4°C

Transform at least 10µL 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 0.05µg/µL Kanamycin

Check the transformed colonies by PCR.

Run the reactions on a gel and select two positive colonies.

Start with 10mL of each colony in LB media with 0.05µg/µL Kanamycin for plasmid isolation.

Isolate plasmids from the two colonies isolated the previous day and elute in 50µL .

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.