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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.
iTUNE
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
iTUNE
FOUNDATIONAL CONCEPTS. PROMOTERS VS. RBS
How are Promoters and RBS related to the CENTRAL DOGMA?
Symbolic representation of a gene expresson unit
iTUNE
FOUNDATIONAL CONCEPTS. PROMOTERS VS. RBS
How are PROMOTERS and RBS determined?
Consensus is built from the pattern found most often in each position
iTUNE
FOUNDATIONAL CONCEPTS. GENE REGULATION
What is the LAC OPERON?
Classic model for inducible gene expression
iTUNE
FOUNDATIONAL CONCEPTS. GENE REGULATION
What is the LAC OPERON?
▸ Elegant genetic architecture consisting of genes for lactose metabolism;
iTUNE
FOUNDATIONAL CONCEPTS. GENE REGULATION
What is the LAC OPERON?
▸ β-galactosidase encoded by lacZ; and
iTUNE
FOUNDATIONAL CONCEPTS. GENE REGULATION
What is the LAC OPERON?
▸β-galactosidase cleaves lactose and its analogs (ONPG).
iTUNE
FOUNDATIONAL CONCEPTS. GENE REGULATION
What is the LAC OPERON?
Regulated by lac repressor protein encoded by lacI.
iTUNE DEVICE WETLAB
PART 2.
iTUNE
EXPERIMENTAL QUESTIONWhich combination of PROMOTER and RBS results in the greatest production of β-galactosidase?
iTUNE
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
iTUNE
▸ 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.
iTUNE
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
iTUNE
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
iTUNE
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
iTUNE
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
iTUNE
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
iTUNE
SHARE YOUR DATA
natbioethics
iTUNE
WHERE CAN IT FIT?
MICROBIOLOGY
ENZYME KINETICS
MOLECULAR GENETICS
WE’RE READY TO ASSAY… ARE YOU?
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