10. 2.4% noble agar: Dissolve 2.4 g of noble agar in Milli-Q H 2 O
and adjust the volume to 100 mL. Autoclave the solution. This
can be stored up to 2 months at 4
C.
11. Solid EMJH plates: 100 mL of 2Â EMJH, 100 mL of 2.4%
noble agar. When preparing the plates, warm the agar solution
in a microwave (with the cap of the agar bottle loosened before
microwaving), and place both solutions in water bath at 56
C.
Next, mix 100 mL of each solution and add 200 μL of sodium
pyruvate (10 mg/mL) and 80 μL of spectinomycin stock solution (see Note 2). Normally, 20 mL of medium are poured into
plates (10 cm diameter).
2.2 Molecular
Biology
1. Thermocycler.
2. Plasmid pMaOri.dCas9 [12].
3. Microtubes, 0.2 mL.
4. PCR reagents (high-fidelity polymerases are preferred) and
PCR product purification kit.
5. UV transilluminator.
6. Dry-heat bath (65
C).
7. T4 DNA ligase and ligation buffer (ATP-containing).
8. E. coli strain π1.
9. Petri dishes with LB medium plus 40 μg/mL spectinomycin
and 0.3 mM dT.
10. Primers pMaOri2F and R:
pMaOri2F: 5
0 ACGCAATGTATCGATACCGAC 3
0 .
pMaOri2R: 5
0 ATAGGTGAAGTAGGCCCACCC 3
0 .
These are flanking primers for the XmaI site we routinely use
for sgRNA inclusion into pMaOri.dCas9.
11. Primer for amplifying lipL32 promoter (restriction site not
included):
p32F: 5
0 GAACAAGAAAGAGTCAGAG 3
0 .
p32R: 5
0 AAAATCACGGTATGAACTTAG 3
0 .
12. Tris-acetate-EDTA (TAE) buffer (10Â): 400 mM Tris,
200 mM acetic acid, 10 mM EDTA. Store at room
temperature.
13. 1% agarose gel: 1% agarose in 1Â TAE buffer.
14. XmaI restriction enzyme.
2.3 Transformation
1. Plasmid pMaOri.dCas9, empty and containing sgRNA cassette
(Fig. 1).
2. L. biflexa serovar Patoc strain Patoc1.
Gene Silencing by Single-Guide RNA and dCas9
111