The BioBuilder Lab Experience: iTune Device

1. The BioBuilder Lab Experience:iTune Device

2. PRESENT • Where can it fit? • Microbiology • Molecular Genetics • Operon Activity • Transcription/Translation • The Big Idea: Evaluate promoter and RBS combinations to optimize β-galactosidase output • Objectives: • Explain the functioning of the lac operon and relate it to this system. • Measure a kinetic chemical reaction. • Culture bacteria using appropriate microbiology techniques. • Properly use synthetic biology and molecular genetics terms. • Enrichment/Extension

3. Design Build Test BioBuilder Emphasis:An Engineering Paradigm The focus of this lab

4. What do we know? And, devices respond to that results in an Parts buildDevices input output An operon is a series of genes that are “coordinately regulated”! Lac Operon is a well-studied operon discovered by Jacob and Monod in 1961

5. What are the Lac Operon Parts? • Consists of 3 genes • LacZ: encodes β-galactosidase • breaks down lactose into glucose and galactose • LacY: encodes lactose permease • membrane protein that facilitates lactose entry • LacA: encodes acetyl transferase • involved in lactose metabolism

6. LacR How Does the Lac Operon Work? Lac Operon Explained • Bacteria prefer glucose, but will eat lactose in a pinch! • Why does this matter? • This system can be ON when lactose is present and OFF when lactose is absent • Can be turned OFFby Lac Repressor protein • Lactose Absent: LacR binds to Operator and prevents transcription • Lactose Present: Lactose binds to LacR allowing transcription RNA Pol RNA Pol

7. Can we use this system to PREDICT and then EVALUATE a device's behavior? Promoters: Strong Medium Weak RBS: Strong Medium Weak LacZ ORF • A: Yes, but not in this form. • Q: Do we have to reconstruct this operon to produce something we can easily see and measure? • A: Yes, the strains you will be testing have been modified to encode LacZ but not LacY and LacA. • Q: What can we measure? • A: β-galactosidase enzymatic activity using different • combinations of Promoters and RBS! • Q: How? • A: ONPG is colorless and similar to lactose. When fed to bacteria, β-gal cleaves it into galactose + O-nitrophenol. This works best at a pH of 7.

8. PREPARATION • Goal: To evaluate promoter and RBS combinations to optimize β-galactosidase output • Advanced Prep... • 1. Streak strains from stabs onto plates. You can view how to prep this here: Streaking from Stabs • 2. Grow strains from plates as liquid overnights. You can view how to prep this here: Liquid Overnight Cultures • 3. Set up McFarland Standards if • Spec 20 is unavailable • 4. Prepare solutions for • β-galactosidase assay: • a. Bicarbonate Buffer • b. ONPG (START) • c. 1M Sodium Carbonate (STOP) • 5. To buy or not to buy... • chloroform??? We’re Ready to Assay... Are you?

9. PERFORM Summary of Protocol: Part 1. (in cuvettes) 1. R, 2-1, 2, or 3//2-4, 5, or 6//2-7, 8, or 9 2. Measure the OD600 of cell dilutions (.9ml buffer + 100µl). Work Flow McFarland Standards 1:10 dilutions OD600 How many cells? Part 2. (in spec tubes) 1. Buffer + undiluted cell sample. 2. Lyse cells with detergent and chloroform (in the hood) 3. START reactions with ONPG at 15 sec intervals 4. STOP reactions with sodium carbonate when yellow 5. Measure absorbance at OD420 for each sample 6. Calculate β-galactosidase activity in Miller Units Add 25 µl undiluted cells 1ml of buffer Add 100 µl detergent and 50 µl chloroform (in hood) OD420 How yellow? Add 1ml sodium carbonate Add 100 µl ONPG *Perform for Blank (no cells), Reference, and 3 other samples

10. Record Data Group Data Class Data

11. Submit Your Data Here: