(100Clg/ml) were added to the media as needed. All cultures were incubated at 37oC for

the appropriate duration.

Recombinant DNA Techniques

 Plasmid DNA was purified with the Qiagen Mini-Prep and Maxi-Prep kits.

Restriction endonuclease digestions, ligations, and transformations were performed

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 Interdisciplinary

Center for Biotechnology Research (ICBR).

Mutagenesis and Strain Construction

 Plasmid pKAM14 (b, Apr) (203) was used to construct all the b subunit mutants.

The mutations were created in each of the plasmids using the Stratagene Quikchange kit.

 Amino terminal mutations. An alanine scan at the amino terminus of the b

subunit, producing basn24ala, thr64ala, glnl04ala, WaS performed by Andrew Hardy, resulting

in the expression of a defective F1Fo ATP synthase complex. Here, the individual sites

were mutated, one at a time, to determine if the defect was the result of any particular

amino acid. Sense and antisense mutagenic oligonucleotides were designed for each of

the desired b subunit mutations (Appendix A) (Figure 5-2A). Three separate site-directed

mutagenesis reactions were performed at codons 2, 6 and 10 of the uncF(b) gene in order

to express mutant b subunits from plasmids pTAM43 (basn24ala), pTAM44 (bthr64ala) and