cells was grown overnight at 37 oC in 5 mL LBG. The overnight culture (100 CIL) was

inoculated into 10 mL pre-warmed (37 oC) LBG and allowed to grow for 2-4 hours. The

fresh culture was poured into sterile polypropylene tubes and incubated on ice for 10

minutes. The bacteria were then harvested by centrifugation (6,000 rpm in a ss-34 rotor)

for 10 minutes. The supernatant was discarded and the tubes were inverted on a

Kimwipe for 1 minute to allow excess LBG to drain. The bacteria pellet was

resuspended in 10 mL cold, sterile 50 mM CaCl2 and incubated on ice for 45-60 minutes.

The bacteria were harvested as described above, resuspended in 1 mL of the 50 mM

CaCl2, and stored at 4 oC until use. Generally, the competent KM2 cells could be used

after two hours on ice but were most efficient for transformation at 24 hours and expired

at 72 hours. For transformation, 100-200 CIL of cells were used as previously described.

 Site-directed mutagenesis. Site-directed mutagenesis was performed either by

means of a Stratagene Quikchange XL kit or by ligation-mediated mutagenesis.

Oligonucleotides containing the desired mutation(s) were designed to anneal to the same

sequence on opposite strands of the plasmid (sense and antisense primers) (Appendix A)

(Figure 2-1). When possible, a silent mutation was encoded to add or delete a restriction

endonuclease recognition sequence to allow for easy screening of the mutation. Primers

were optimally designed by ensuring the mutation was in the middle of the sequence, a

cytosine (C) or guanine (G) flanked both ends of the sequence, and the melting

temperature (Tm) was greater than or equal to 78 oC. The Tm was calculated as

Tm=81.5+0.41 (%GC)-675/N-%mismatch, where N was the primer length (bases). When

introducing insertions or deletions, "%mismatch" was dropped from the formula.