The BioBuilder Lab Experience:iTune Device

Present Prepare Perform

PRESENTThe 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

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BioBuilder Emphasis:An Engineering Paradigm

Design Build

Test The focus The focus of thisof this lablab

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 aninput output

What are the Lac Operon Parts?

Consists of 3 genesLacZ: encodes β-galactosidase

breaks down lactose into glucose and galactoseLacY: encodes lactose permease

membrane protein that facilitates lactose entryLacA: 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 RNA PolPol

RNA RNA PolPol

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:Promoters:StrongStrong

MediumMediumWeakWeak

RBS:RBS:StrongStrong

MediumMediumWeakWeak

LacZ LacZ ORFORF

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PREPARATIONGoal: To evaluate promoter and RBS combinations to

optimize β-galactosidase outputAdvanced 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 Cultures3. Set up McFarland Standards if Spec 20 is unavailable4. Prepare solutions for β-galactosidase assay:

a. Bicarbonate Bufferb. 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 intervals4. STOP reactions with sodium carbonate when yellow5. Measure absorbance at OD420 for each sample6. 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

OD420How 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).

Sample Strain Abs 600 Start time Stop time Time elapsed Abs 420 b-gal activity

B = blank none 0:00

R reference 0:15

1 (weak) 2-1, 2, or 3 0:30

2 (medium) 2-4, 5, or 6 0:45

3 (strong) 2-7, 8, or 9 1:00

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