Transcript - BioBuilder

The BioBuilder Lab Experience:
iTune Device
Present
Prepare
Perform
PRESENT
Where can it fit?
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.
Microbiology
Molecular Genetics
Operon Activity
Transcription/Translation
Enrichment/Extension
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are needed to see this picture.
Properly use synthetic biology and
molecular genetics terms.
QuickTime™ and a
GIF decompressor
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BioBuilder Emphasis:
An Engineering Paradigm
Design
Build
Test
The focus of
this
lab
What do we know?
Parts build Devices
And, devices respond to
input that results in an output
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 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!
Why does this matter?
This system can be ON when lactose is present and OFF when
lactose is absent
Can be turned OFF by Lac Repressor protein
LacR
Lactose Absent: LacR binds to Operator and prevents transcription
Lactose Present: Lactose binds to LacR allowing transcription
RNA Pol
RNA Pol
Can we use this system to PREDICT and then
EVALUATE a device's behavior?
Promoters:
Strong
Medium
Weak
RBS:
Strong
Medium
Weak
LacZ
ORF
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.
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PREPARATION
Goal: To evaluate promoter and RBS combinations to optimize βgalactosidase output
Advanced 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 Cultures
We’re Ready to Assay... Are you?
3. 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???
PERFORM
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
McFarland Standards
Work Flow
1:10
dilutions
OD600
How many cells?
dilutions (.9ml buffer + 100µl).
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
1ml of
buffer
Add 25 µl
undiluted
cells
Add 100 µl
detergent and
50 µl chloroform
(in hood)
OD420
How yellow?
Add 100 µl
ONPG
Add 1ml
sodium
carbonate
*Perform for Blank (no cells),
Reference, and 3 other samples
Record Data
Group Data
Sample
Strain
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
Class Data
Abs 600
Start time
1:00
Stop time
Time elapsed
Abs 420
b-gal activity
Submit Your Data Here:
http://www.biobuilder.org/activities/
Password: natbioethics