Assay for an Antibiotic Resistance Enzyme

1. Assay for an Antibiotic Resistance Enzyme Carolina Kit Focus Color Assays, pH

2. Background Info. • What is an Enzyme • There are many types of enzymes and each is assigned a specific task. Enzymes are basically proteins which bring about a chemical reactions to maintain balance in body functions. • In this lab the enzymes being used are antibiotic resistance enzymes produced by ampicillin-resistant bacteria.

3. Background Info. • Antibiotic resistance in bacteria • Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic.

4. Background Info. • Genes that confer resistance can easily be transferred between bacteria, through conjugation, transduction, or transformation. • Video

5. Background Info. • Enzyme assays • Are Laboratory methods or tests for measuring enzymatic activity • Colorimetric assays • Utilizing color/color change in order to see enzymatic activity

6. Timeline/Overview • Monday—Lecture • Tuesday—pre-lab quiz, Part 1 • Thuesday--Part 2 (volunteer to serve as demonstrator—4 hours lab service) • Monday--handouts due, post quiz

7. Prep for Lab. • 2 days prior --label tubes control (8--15mL tubes) pAMP (8--15 mL tubes) penicillin-G (8--15 mL tubes) --streak fresh plate of MM294 and MM294/pAMP (24 hours at 37 incubator) • 1 day prior --prepare cultures (1/group)—see teacher guide (24 hours at 37 incubator) --penicillin-G solution (store at -20 until use) --aliquot (8 groups) • MM294 5ml overnight culture, label control • MM294/pAMP 5ml overnight culture, label pAMP • 5ml of 4mg/mL penicillin-G • On lab day 1 --test the reagents with phenol red • Lab day 2 --demonstrator of spectrometer

8. Overview and tips(Tuesday) • Use correct plate streaking technique • Make sure incubation is set to 37 degrees C.

9. Overview and tips • When inoculating use Aseptic technique • Don’t forget to check the temperature Should be set to 37 degrees C