The BioBuilder Lab Experience: iTune Device PresentPreparePerform

PRESENT 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. Where can it fit? Microbiology Enrichment/Extension Molecular Genetics Operon Activity Transcription/Translation

BioBuilder Emphasis: An Engineering Paradigm DesignBuild Test The focus of this lab lab

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 What do we know? build DevicesParts And, devices respond to that results in an inputoutput

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

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

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. Can we use this system to PREDICT and then EVALUATE a device's behavior? Promoters:StrongMediumWeak RBS:StrongMediumWeak LacZ ORF

PREPARATION Goal: To evaluate promoter and RBS combinations to optimize β- galactosidase output Advanced Prep Streak strains from stabs onto plates. You can view how to prep this here: Streaking from Stabs Streaking from Stabs 2. Grow strains from plates as liquid overnights. You can view how to prep this here: Liquid Overnight Culturestures 3. Set up McFarland3. 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?

Add 1ml sodium carbonate PERFORM 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 Work Flow *Perform for Blank (no cells), Reference, and 3 other samples McFarland Standards 1:10 dilutions OD600 How many cells? 1ml of buffer Add 100 µl detergent and 50 µl chloroform (in hood) Add 100 µl ONPG Add 25 µl undiluted cells OD420 How yellow? 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 ).

SampleStrainAbs 600Start timeStop time Time elapsed Abs 420 b-gal activity B = blanknone0:00 Rreference0:15 1 (weak)2-1, 2, or 30:30 2 (medium)2-4, 5, or 60:45 3 (strong)2-7, 8, or 91:00 Record Data Group Data Class Data

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