Difference between revisions of "Team:UCLouvain/Protocols"

This protocol is adapted from Jiang et al., 2015. It assumes a version of pTargetTet targeting the correct locus of the E. coli genome has already been created, as well as a donor DNA that include overlap regions and the gene to be inserted. For information on the design and creation of the sgRNA-producing pTarget, see Selection of the sgRNA and modification of pTarget.

• E. coli strain containing the pCas plasmid.
• pTargetTet targeting the locus to be modified.
• Donor DNA: gene of interest flanked by two 500-bp overlaps, or an ssDNA for deletion.
• LB, Kanamycin, Tetracyclin.
• Arabinose 10%.
• Chilled sterile ddH2O.
• Sterile PEP tube.
• IPTG 0,1 M.

pCas has a thermo-sensitive origin of replication. All steps until pCas curing should be incubated at 30°C.

Day 1

1. Start a 5 ml pre-culture of TOP10 pCas in LB-Kanamycin. Incubate it overnight at 30°C without shaking.

Day 2

Competent cells

1. Start a culture for transformation by inoculating 40 ml of LB-Kan with 400 µl of pre-culture. Incubate in a 250 ml flask at 30°C until an OD600 between 0,4 and 0,6 is attained.
2. Add 600 µl of 10% arabinose to the culture to cause the induction of the λ-Red genes. Incubate for 15 minutes at 30°C.
3. Chill the culture in a water-ice bath. Transfer to a 50 ml centrifuge tube and centrifuge at 4600 x g for 7 minutes in a centrifuge cooled to 4°C.
4. Add 30 ml of ice-cold water to the pellet. Swirl gently to resuspend the cells. Centrifuge again as described in step 5.
5. Resuspend the pellet in 1 ml of cold ddH2O. Transfer it using a 1000 µl tip to a pre-chilled sterile EP tube. Centrifuge down at 10.000 x g for 4 seconds in a chilled tabletop centrifuge. (Note, due to speed-up time, doing a quickspin up to 10.000 x g and then letting it slow down is usually good enough) Carefully pipette out the supernatant.
6. Repeat step 7.
7. Resuspend cells in 200 µl of chilled ddH2O. Keep on ice until ready to transform.

Transformation

1. Using a 1-mm transformation culture, electroporate 100 ng of pTargetTet plasmid and 400 ng of linear donor DNA in 50 µl of competent cells (Note: when using an ssDNA oligo instead of dsDNA linear donor DNA, transform 100 ng of the plasmid with 100 ng of oligo). Recover the cells at 30°C for 1 hour in SOC medium then plate on LB-Tet-Kan plates. Incubate the plate at 30°C overnight. (Note: it often takes up to 48 hours for colony to show up).
2. Screen transformant by colony PCR to identify correct clones.

Plasmid curing

1. To cure the plasmids, resuspend a colony in 2 ml of LB-Kan to which 10 µl of IPTG 0,1 M are added (Final concentration: 0,5 mM) and the culture is grown overnight at 30 °C. In the morning, the colonies are plated on LB plates and incubated at 37°C O/N to cure them of the pCas plasmid.

• 400 W LEO / S-400-94-CR light
• Five 12-wells plates
• M9, LB
• Tyrosine, ortho-nitrobenzyl tyrosine, L-arabinose

1. Inoculate 10 mL of liquid LB and incubate at 37°C overnight with appropriate antibiotic
2. Bl21(DE3), TyrA- (not transformed), TyrA- + RFP

1. Pellet precultures at 3000 xg for 10 minutes at 4°C
2. Wash 3x with 5 mL liquid M9 to remove tyrosine present in medium
3. When OD600 ~ 0.6, concentrate 10X
4. Add the following in each well of a 12-wells plate (final volume 3 mL, completed withliquid M9). Make three replicas.

Tyr + Ara (BL21)	Tyr (BL21)	ONB-Tyr + Ara (BL21)	ONB-Tyr (BL21)
Tyr + Ara (TyrA-)	Tyr (TyrA-)	ONB-Tyr + Ara (TyrA-)	ONB-Tyr (TyrA-)
Tyr + Ara (TyrA-/RFP)	Tyr (TyrA-/RFP)	ONB-Tyr + Ara (TyrA-/RFP)	ONB-Tyr (TyrA-/RFP)

• Tyrosine: 0.05 mg/mL
• ONB-Tyr: 0.084 mg/mL
• L-arabinose: 0.1% (w/v)
• KAN30, CM34