according to the recommendations of the manufacturers (New England Biolabs,

Stratagene and Life Technologies, Inc.). Site-directed mutagenesis was performed either

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

fragments were separated in 0.8 % agarose gel by electrophoresis and purified using a

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

 Plasmid pKAM14 (b, Apr) (203) was used to construct b subunits with a deletion of

the last four amino acids. Plasmids pKAM14 (b, Apr) (203), pTLC11 (barg364ile, Apr), or

pTLC 15 (barg364glu, Apr) (215) were used to construct the epitope tagged b subunits.

Epitope tags were inserted into each of the plasmids using the Stratagene Quikchange kit.

A four amino acid carboxyl-terminal truncation was accomplished by deletion of the final

four codons of the uncF(b) gene to express bal534end (Appendix A) (Figure 3-1A). The

restriction endonuclease recognition sequence for HindlII was constructed near the

deleted sequence for an initial detection of the truncation. A histidine epitope tag was

inserted at the N-terminus by mutagenesis between the first and second codons of the

uncF(b) gene to express bhis, bAl534end-his or b+124-130-his (Figure 3-1B). All of the

recombinant histidine-tagged b subunit plasmids were then digested with PstI and Ndel

and subsequently ligated into a plasmid conferring the chloramphenicol resistance gene

and the pACYC 184 origin of replication (Table 3-1). A V5 epitope tag was added to the

C-terminus by site-directed mutagenesis before the termination codon of the uncF(b)

gene to express bV5, barg364ile-V5, Or barg364glu-V5 (Figure 3-1B). The recombinant V5-

tagged b subunit plasmids included the ampicillin resistance gene and the pUC18 origin