THE BIOBUILDER LAB EXPERIENCE: iTUNE DEVICE BioBuilder emphasizes an

engineering paradigm…

Repeated iterations of the

Design, Build, Test Cycle in

each BioBuilder module

Build

Test

Design

Test

BioBuilder Emphasis

iTUNE

OBJECTIVESLET’S�TAKE�A�CLOSER�LOOK!

▸ To TEST several variants of an enzyme-generating genetic circuit;

▸ To MEASURE circuits’ outputs by using an enzymatic assay; and

▸ To EXPLORE the importance of engineering principles enabling functional assembly.

INTRODUCTION TO iTUNE DEVICE

PART 1.

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WHAT DO WE KNOW?▸ Synthetic biology seeks to

customize a playlist of GENETIC PARTS to generate precise behaviors;

▸ BEHAVIORAL SEPARATION between the operation of parts to minimize unanticipated interactions is a challenge; and

▸ MEASUREMENT is useful in describing, controlling, assembling, and improving what is being measured.

MODULARITY

INSULATION

1 smoot = 1.70m

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FOUNDATIONAL CONCEPTS. PROMOTERS VS. RBS

How are Promoters and RBS related to the CENTRAL DOGMA?

Symbolic representation of a gene expresson unit

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FOUNDATIONAL CONCEPTS. PROMOTERS VS. RBS

How are PROMOTERS and RBS determined?

Consensus is built from the pattern found most often in each position

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FOUNDATIONAL CONCEPTS. GENE REGULATION

What is the LAC OPERON?

Classic model for inducible gene expression

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FOUNDATIONAL CONCEPTS. GENE REGULATION

What is the LAC OPERON?

▸ Elegant genetic architecture consisting of genes for lactose metabolism;

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FOUNDATIONAL CONCEPTS. GENE REGULATION

What is the LAC OPERON?

▸ β-galactosidase encoded by lacZ; and

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FOUNDATIONAL CONCEPTS. GENE REGULATION

What is the LAC OPERON?

▸β-galactosidase cleaves lactose and its analogs (ONPG).

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FOUNDATIONAL CONCEPTS. GENE REGULATION

What is the LAC OPERON?

Regulated by lac repressor protein encoded by lacI.

iTUNE DEVICE WETLAB

PART 2.

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EXPERIMENTAL QUESTIONWhich combination of PROMOTER and RBS results in the greatest production of β-galactosidase?

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EXPERIMENTAL QUESTIONWhich combination of PROMOTER and RBS results in the greatest production of β-galactosidase?

You will be comparing 9 GENE REGULATORY DESIGNS

constructed to bear a single promoter:RBS:lacZ unit

against a REFERENCE

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▸ Prepare MCFARLAND TURBIDITY STANDARDS to measure cell density (if needed)

0 1 2 3 4 5 6 7

1% BaCl2 + 1% H2SO4

LAB PROTOCOL. ADVANCED PREP.

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Day 2. Grow liquid overnights of 10 bacterial strains (3ml LB + 3µl amp + 30µl IPTG to relieve inhibition of lacZ)

Day 1. Streak strains from stabs onto LB/amp plates

LAB PROTOCOL. ADVANCED PREP. LAB PROTOCOL. WORK FLOW.iTUNE

▸ Part 1. CELL DENSITY

1. Make 3ml of 1:10 dilution of each sample (300 µl cells + 2.7 ml bicarbonate buffer).

2. TRANSFER diluted mixture to cuvettes.

3. If a spectrophotometer is available, MEASURE the absorbance at OD600. Record this value TIMES 10 as the density of the undiluted sample. Or, use McFarland Standards.

BUFFER

2-R 2-1 2-4 2-7

LIQUID OVERNIGHTS UNDILUTED

YOUR SAMPLES

OD600

2-R 2-1 2-4

LIQUID OVERNIGHTS 1:10 DILUTIONS

2-7

YOUR SAMPLES

McFarland Standards

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LAB PROTOCOL. WORK FLOW

1. Add 1ml bicarbonate buffer to 4 test tubes.

▸ Part 2. ENZYME ASSAY

BUFFER

2. Add 100µl undiluted culture to each reaction tube.

2-R 2-1 2-4 2-7

REACTION TUBES

YOUR SAMPLES

3. LYSE the cells by adding 100µl of lysis solution (you will make this). Vortex for 10”.

4. Add 100µl of ONPG to the first tube to START the reactions. Wait 15” and add 100µl of ONPG to the next tube. Repeat.

5. After 10’ STOP the reactions by staggering the addition of 1ml of soda ash/sodium carbonate.

Na2CO3

2-R 2-1 2-4 2-7

LIQUID OVERNIGHTS UNDILUTED

YOUR SAMPLES

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LAB PROTOCOL. DATA COLLECTION.

2. Read the absorbance of each sample at OD420. OD420

3. Calculate the β-galactosidase activity in Miller units for each sample using the formula.

β-gal production in Miller Units:

___Abs420____ (t * v * Abs600) 1000 *

t = time (minutes) v = volume of cells added to reaction in mL

1. Transfer some of the reaction mixture to a cuvette. 2-R 2-1 2-4 2-7

REACTION TUBES

Blank

YOUR SAMPLES

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LAB PROTOCOL. RECORD YOUR DATA.Strain Promoter RBS Abs 600 Start Time Stop Time Time Elapsed

(min)Abs 420

B none none 0:00

2-R Reference Promoter

Reference RBS 0:15

2-1 weak weak 0:30

2-2 weak medium 0:45

2-3 weak strong 1:00

2-4 medium weak 1:15

2-5 medium medium 1:30

2-6 medium strong 1:45

2-7 strong weak 2:00

2-8 strong medium 2:15

2-9 strong strong 2:30

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LAB PROTOCOL. RECORD YOUR DATA.Strain Promoter RBS β-gal Activity in Miller units Class Mean

2-R Reference Promoter

Reference RBS

2-1 weak weak2-2 weak medium2-3 weak strong2-4 medium weak2-5 medium medium2-6 medium strong2-7 strong weak2-8 strong medium2-9 strong strong

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SHARE YOUR DATA

natbioethics

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WHERE CAN IT FIT?

MICROBIOLOGY

ENZYME KINETICS

MOLECULAR GENETICS

WE’RE READY TO ASSAY… ARE YOU?