Qiagen, Inc. QIAquick Gel Extraction kit. Plasmid sequences were determined by

automated sequencing in the core facility of the University of Florida ICBR.

Mutagenesis and Strain Construction

 Plasmids pKAM14 (b, Apr) (203), pAUL5 (ball, Apr) (194) or pAUL47 (b.11, Apr)

(193) and pJLG1 (a, c, b, Cmr) (167) were used to construct cysteine mutant b subunits.

Mutagenesis was performed in three different subunits; therefore, the ultimate goal was

to express the entire unc operon containing the desired cysteine mutations from one

plasmid. Plasmid pJLG1 (a, c, b, Cmr) was used as a temporary vector in order to

facilitate cloning due to its extra unique restriction enzyme sites.

 b subunit mutations. In order to construct the b subunit mutations, plasmids

pAUL5 (ball, Apr), pAUL47 (b it, Apr) and pJLG1 (a, c, b, Cmr) were first digested with

restriction enzymes PpuM~I and Narl in order to move the b subunit deletion and insertion

into the pJLG1 plasmid. The resulting 204 and 270 bp cassettes isolated from pAUL5

(ball, Apr) and pAUL47 (b.11, Apr), respectively, were subsequently ligated into the

pJLG1 vector, resulting in pTAM1 (ball, Cmr) and pTAM3 (b it, Cmr) (Table 4-1). In

order to site specifically label the b subunit in future experiments, a native amino acid

was mutated to a cysteine. Two sites were chosen for replacement, bser84 and bgly43,

located respectively above and below the region of length alteration. Furthermore, the b

subunit had a native cysteine, bcvs20, lOcated in the membrane-spanning portion of the

subunit, which was mutated to a serine. The mutations were created in each of the

plasmids using the Stratagene Quikchange kit. Sense and antisense mutagenic

oligonucleotides were created for each of the desired b subunit mutations (Appendix A)

(Figure 4-2A). First, the native cysteine was abolished by mutagenesis of codon 20