Team:Florida/Experiments
Experiments
Workflow
The following procedure was repeated for all three components (SCRIBE, Cas9, pkdtrpoB565).
1. PCR amplification
2. Gel -> Clean-up (Miniprep) -> Dpn1: recognizes methylation sites from wild types
3. Cloning CPEC
4. Transformation + plating
5. Colony PCR to check
6. Work with plasmid with E. coli
PCR amplification:
Make solution of...
1. Template: Cell Lysis (thermocycler) 98°C for 5 min
2. Nanopure water
3. Mastermix (Q5)
4. Primers
5. dnTPs
6. Magnesium
7. Buffer
8. Annealing temperature: 49°C
Gel Electrophoresis
1. Mix PCR products with dye
2. Put ladder into gel
3. Put the mixture into the wells
4. Run gel at 100 volts, 30 minutes
PCR Clean-up Miniprep
Ethanol Purification
1. Add 1uL Sodium Acetate
2. Sit in -20 °C overnight
3. Spin for 15 minutes to get DNA at bottom
4. Wash with 110uL 70% ethanol
5. Resuspend in EB
6. Place in -80°C
Column Purification
1. 1.5mL of each colony culture into tube
2. Centrifuge at max for 1 minute
3. Add another 1.5mL into each tube
4. Centrifuge at max speed for 1 minute
5. Resuspend pellet in 250uL buffer P1 and transfer to a microcentrifuge tube
6. Add 250uL of P2 mix thoroughly by inverting 4-6 times until solution becomes clear
7. Add 350uL of N3 and mix immediately by inverting.
8. Centrifuge for 10 min at 13,000 rpm
9. Apply supernatant from step 5 to Q1A prep spin column by pipetting. Centrifuge for 30-60 seconds & discard flow through the Q1A prep column and swittch off the vacuum source.
10. Add 500uL of PB Buffer
11. Wash spin column by adding 750uL PE buffer. Centrifuge for 30-60 seconds and discard flow. Transfer Q1A spin column to collection tube.
12. Centrifuge for 1 minute and remove residual wash buffer.
13. Place Q1A spin column in clean microcentrifuge tube.
14. To elute DNA, add 50uL EB buffer to column. Let it sit for 1 minute and centrifuge for 1 minute.
Cloning (CPEC)
1. Anneal piece
2. Put CPEC (4uL ) into DH-alpha cells (Wait 30 min)
3. Water bath (45 seconds) 42°C
4. 2 minutes on ice
5. Add SOB
6. Recover for 1 hour (37°C incubator)
7. Plate on LBkan50 plate
Transformation
1. Spin to get DNA to bottom of tubes
2. Add 1uL of plasmid A & B into other two tubes with cells (E. coli dHα)
3. Place 30 min on ice.
4. 42°C: heat shock in water bath for 45 sec
5. Add to ice for 2 min
6. Add 1 mL of SOB.
7. Incubate at 37°C for 1 hour
8. Spread culture using beads
9. Pipette 100uL onto plate
10. Spin tube for pellet
11. Re-suspend in media
12. Transfer liquid to plates
13. Incubate plates at 37°C
Electroporation
Electroporation: apply electrical pulse so that it pokes holes in the cell membrane. DNA can now enter the cell through those pores.
1. Water has other charges that will cause electrons to bind to them instead of cell membrane Make cells electrocompetent in order to complete electroporation
2. Add 1.5 mL BL21 to tube on ice
3. Spin at 4°C at 5 speed for 2 minutes to pellet the cells
4. Extract supernatant
5. Resuspend pellet in 500 uL cold nanopure, 500 uL glycerol mannitol
6. Spin for 2 minutes, vacuum liquid
7. Resuspend in 50 uL glycerol mannitol
8. Transfer to cuvette
9. Electroshock cuvettes
10. Suspend in SOB, transfer to tubes, incubate
Colony PCR
-100 uL water into each well
-Put colonies into each well. Out tip into test tube with 5 mL SOB and 5 uL Kan
-Pick up 6 uL of water w colonies and put into the second strip of wells
Serial Dilutions
A. Culturing Tubes
1. 5mL per tube of LB
2. 5uL of each antibiotic and IPTG (chlor40, spec100, kan50, and 0.1 mM IPTG)
3. Use stick to pick up colony and place in tubes
4. Put in 30°C incubator.
B. Plating
0 hr (no ATC)
10-3 to 10-8 dilution on non-selection plate
0 hours (3 replicates)
1st hour (3 replicates)
2nd hour (3 replicates)
3rd hour (3 replicates)
Comparing Rifampicin, LB, and Triple Antibiotics plates
1. Adding 180uL of H2O into each well of 96 well plate
2. Add 20uL of culture in first 3 wells
3. Add 20 uL from previous well into the one after to make 10^-1, 10^02 dilution, etc all the way up to 10^-6
4. Diluted 3 times in well plate
5. Place 10uL of each corresponding dilution onto all 6 plates using multichannel pipette.
Incorporating rpoB in SCRIBE
Firstly, we replaced the original target sequence on the SCRIBE plasmid pFF745 with the rpoB target sequence. Primers 2105, 2106, 2107, and 2108 were added to conduct a PCR amplification in which everything on the plasmid excluding the target sequence were amplified. Then, using Circular Polymerase Extension Cloning (CPEC), the rpoB sequence was attached to the pFF745 plasmid because the ends were complementary. The rpoB was obtained through purchase and contains some mutations on the sequence that will be transcribed later by the host E. coli cells. In this state, the plasmid is single stranded so Q5 master mix was utilized for PCR amplification in order to make the plasmid double stranded. We used Q5 because of its low error prone rate (1 in 1,000,000). Now, the plasmids are ready to be transformed into competent DH alpha E.coli cells. The cells were given a day to recover and then plated on agar plates with the corresponding antibiotic resistance: chloramphenicol. To distinguish between the cells with and without the new insert, colony PCR and gel electrophoresis was carried out. The forward primer binds to the target sequence while the reverse primer binds to the backbone. The forward primer only corresponds to rpoB therefore only cells with the new insert should show the corresponding band when performing gel electrophoresis. Lastly the cells with SCRIBE were induced with IPTG. rpoB encodes for the B subunit of the polymerase and contains mutations on the sequence that will be permanently incorporated into the E. coli cells through the SCRIBE system to give them rifampicin resistance. To test that SCRIBE, we plated the cultures induced with IPTG on rifampicin plates. If the cells grew, it was concluded that the SCRIBE system was working in the cells.
Incorporating Cas9 and PkdtrpoB565
For the next part, we are incorporating the CRISPR/Cas9 system onto separate plasmids to also be incorporated in the DH-alpha cells containing the SCRIBE system using electroporation. At first, we planned to incorporate the SCRIBE system, Cas9, and pkdtrpoB565 all on one plasmid but the cloning was the transformation portion was unsuccessful. At most, some cells contained only SCRIBE and Cas9 or SCRIBE and the guide RNA. To decipher which colonies had which combination we plated the cells containing kan/spec or kan/chlor. Then we transformed each cells with the missing component. We adjusted the procedure for additional replications to have each component cloned on separate plasmids.
The goal of this part of the project was to see whether the CRISPR/Cas9 system was as effective as the rifampicin antibiotic selection marker.
Source: Redman M, King A, Watson C, et alWhat is CRISPR/Cas9?Archives of Disease in Childhood - Education and Practice 2016;101:213-215.
We altered the promoter next to the reverse transcriptase component of the pFF745 to the promoter J23101. J23101 is a promoter synthetically designed to give high expression of the reverse-transcriptase sequence as well as the β-subunit sequence. The PkdtrpoB565 sequence was incorporated into the pFF745 plasmid and then transformed into the dH-alpha cells first. PkdtrpoB565 has spectinomycin resistance and encodes the guide sequence which binds complementary to the wild type sequence. Next, the Cas9 sequence was incorporated into the cells to probe for guide RNA bound to the wild type colonies. Cas9 then makes lethal double-stranded breaks to kill the wild types, leaving only the mutant colonies growing on the plates.
Note: Because both Cas9 and SCRIBE have chloramphenicol resistance, the SCRIBE plasmid was altered to have kanamycin resistance in order to differentiate between the three components.
Under the CRISPR/Cas9 system, ATC induces the tetracycline promoter which activates Cas9. The cells were split between plates with ATC and plates without ATC. Then for each set we made serial dilutions for the cells on plates with the three antibiotics (kan, spec, chlor), LB, and rifampicin to compare the efficiency rates based on plate count.