The BioBuilder Lab Experience: iTune Device
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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
Record DataGroup Data
Class Data
Submit Your Data Here:
http://www.biobuilder.org/activities/Password: natbioethics
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