Team:Michigan/Experiments

Building the Attack/Guard Assay:

SaCas9, gRNA for SaCas9, and gRNA for SpCas9 were synthesized by Integrated DNA Technologies. SaCas9 was synthesized from two linearized fragments, with a Ava restriction site near the 5’ end of one and 3’ end of the other to join them into one fragment. This complete fragment was flanked by EcoRI and PstI sites in order to be compatible for insertion into the submission vector pSB1C3. Once the SaCas9_C plasmid was confirmed, we double digested the SaCas9_C and the gRNA for SaCas9 with SacI and SgrAI and then ligated the fragments, leaving us with one of our final plasmids needed for experiments, SaCas9_C_gRNA, a plasmid containing a LacI coding region for inducible control, the gRNA able to transcribe and bind with SaCas9 in order to target the complementary sequence on the reporter plasmid, and the SaCas9 coding region. We obtained the SpCas9 DNA from the Zhang lab at the University of Michigan in a pET083 backbone. In order to move the fragment we needed (containing LacI repressor and SpCas9) into pSB1K3, we used PCR mutagenesis to flank our region of interest with EcoRI and PstI cut sites. Once SpCas9_K was confirmed, we double digested with the gRNA for SpCas9 using sites EcoNI and SgrAI. Then we ligated the resulting fragments, leaving us with our final plasmid needed for experiments, SpCas9_K_gRNA, a plasmid containing a LacI Repressor coding region for inducible control, the gRNA able to transcribe and bind with SpCas9 in order to target the complementary sequence on the reporter plasmid, and the SpCas9 coding region.

Given more time to conduct experiments, we would induce the necessary point mutations in each Cas9 through the use of One-Pot Mutagenesis (see protocol section). This requires primers be designed to anneal to the specific portion of the the sequence where we want to induce our mutations. These mutations will be specific to each Cas9 used since the nuclease domain will be in a different location within the sequence in each case.

The reporter plasmid (with CFP) was synthesized by Integrated DNA Technologies to contain the necessary target sequence and PAM sequence needed for recognition of SpCas9 and SaCas9 with their respective gRNAs.

All restriction digests were performed using New England Biolabs (NEB) restriction enzymes. Qiagen PCR cleanup kits were then used on each restriction digest product. All ligations were performed with NEB T4 ligase at room temperature overnight, then the ligation products were first transformed into homemade DH5a chemically competent cells (see protocol section), but after a couple failed transformations, we used NEB DH5a chemically competent E. coli cells. These transformations were plated and grown overnight. 5mL liquid cultures were prepared from colonies picked from the plates the next day and themselves grown overnight. All DNA extraction was performed using Qiagen miniprep kits. CFP was measured at 398 nm excitation and 498 nm emission via a plate reader (Infinite 200, Techan Life Sciences). Cell density was measured at A600 via the same plate reader.

Protocols

Experiment 1:

Will SpCas9 + Reporter and SaCas9 + Reporter effectively degrade the Reporter plasmid, reducing CFP over time?
Setup:
Start with 3 types of 5mL liquid cultures grown overnight shaking 225 rpm at 37C: Reporter plasmid (CFP - Amp), SpCas9 with gRNA and Reporter with target sequence (Kan + Amp), and SaCas9 with gRNA and Reporter with target sequence (Chlor + Amp).

1. Add 4mL prewarmed (37C) LB-Amp (2 15mL confocal tubes), add 4mL prewarmed (37C) LB-Kan + Amp (3 15mL confocal tubes), and add 4mL prewarmed (37C) LB-Chlor + Amp (3 15mL confocal tubes)
2. Add 1mL from each 5mL starter culture to three 4 mL of prewarmed media for SpCas9 and SaCas9 (plus the Reporter CFP plasmid for each) and two 4 mL of prewarmed media for the Reporter CFP plasmid.
3. Grow all 8 5mL cultures at 37C until A600 is between 1.0 and 1.2.
4. When all cultures are between 1.0 and 1.2, record A600 for each culture (Time 0), measure CFP and image fluorescence.
5. Add IPTG to a final concentration of 1 mM to two cultures of SpCas9 and SaCas9 and to one tube of the Reporter plasmid.
6. Every 15 minutes take measurements of A540 for all cultures, measure CFP (via plate reader), and image fluorescence for 1 hour.
7. Every 30 minutes take measurements of A540 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.
8. Every 1 hour take measurements of A540 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.

Experiment 2:

Will dSpCas9 + Reporter and dSaCas9 + Reporter result in little to no changes in CFP level compared to control?
Setup:

Start with 3 types of 5mL liquid cultures grown overnight shaking 225 rpm at 37C: Reporter plasmid (CFP - Amp), dSpCas9 with gRNA and Reporter with target sequence (Kan + Amp), and dSaCas9 with gRNA and Reporter with target sequence (Chlor + Amp).
1. Add 4mL prewarmed (37C) LB-Amp (2 15mL confocal tubes), add 4mL prewarmed (37C) LB-Kan + Amp (3 15mL confocal tubes), and add 4mL prewarmed (37C) LB-Chlor + Amp (3 15mL confocal tubes)
2. Add 1mL from each 5mL starter culture to three 4 mL of prewarmed media for dSpCas9 and dSaCas9 (plus the Reporter CFP plasmid for each) and two 4 mL of prewarmed media for the Reporter CFP plasmid.
3. Grow all 8 5mL cultures at 37C until A600 is between 1.0 and 1.2.
4. When all cultures are between 1.0 and 1.2, record A600 for each culture (Time 0), measure CFP and image fluorescence.
5. Add IPTG to a final concentration of 1 mM to two cultures of dSpCas9 and dSaCas9 and to one tube of the Reporter plasmid.
6. Every 15 minutes take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 1 hour.
7. Every 30 minutes take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.
8. Every 1 hour take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.

Experiment 3:

What are the results with E.Coli that have been transformed with SpCas9, dSacas9, and Reporter?
Setup:
Start with 2 types of 5mL liquid cultures grown overnight shaking 225 rpm at 37C: Reporter plasmid (CFP - Amp), SpCas9 with gRNA, Reporter with target sequence (Kan + Amp), and dSaCas9 with gRNA (Chlor + Amp + Kan).
1. Add 4mL prewarmed (37C) LB-Amp (2 15mL confocal tubes) and add 4mL prewarmed (37C) LB-Chlor + Amp + Kan (4 15mL confocal tubes)
2. Add 1mL from each 5mL starter culture to four 4 mL of prewarmed media for E.coli containing SpCas9, dSaCas9, and reporter plasmids and two 4 mL of prewarmed media for the Reporter CFP plasmid.
3. Grow all 6 5mL cultures at 37C until A600 is between 1.0 and 1.2.
4. When all cultures are between 1.0 and 1.2, record A600 for each culture (Time 0), measure CFP and image fluorescence.
5. Add IPTG to a final concentration of 1 mM to two cultures of the E.coli containing SpCas9, dSaCas9, and reporter plasmids and to one tube of the Reporter plasmid.
6. Every 15 minutes take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 1 hour.
7. Every 30 minutes take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.
8. Every 1 hour take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.

Experiment 4:

What are the results with E.Coli that have been transformed with SaCas9, dSpCas9, and Reporter?
Setup:
Start with 2 types of 5mL liquid cultures grown overnight shaking 225 rpm at 37C: Reporter plasmid (CFP - Amp), dSpCas9 with gRNA, Reporter with target sequence (Kan + Amp), and SaCas9 with gRNA (Chlor + Amp + Kan).
1. Add 4mL prewarmed (37C) LB-Amp (2 15mL confocal tubes) and add 4mL prewarmed (37C) LB-Chlor + Amp + Kan (4 15mL confocal tubes)
2. Add 1mL from each 5mL starter culture to four 4 mL of prewarmed media for E.coli containing dSpCas9, SaCas9, and reporter plasmids and two 4 mL of prewarmed media for the Reporter CFP plasmid.
3. Grow all 6 5mL cultures at 37C until A600 is between 1.0 and 1.2.
4. When all cultures are between 1.0 and 1.2, record A600 for each culture (Time 0), measure CFP and image fluorescence.
5. Add IPTG to a final concentration of 1 mM to two cultures of the E.coli containing dSpCas9, SaCas9, and reporter plasmids and to one tube of the Reporter plasmid.
6. Every 15 minutes take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 1 hour.
7. Every 30 minutes take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.
8. Every 1 hour take measurements of A600 for all cultures, measure CFP (via plate reader), and image fluorescence for 2 hours.

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