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The BioBuilder Lab Experience:
Eau That Smell!
Present
Prepare
Perform
PRESENT
Where can it fit?
The Big Idea: Compare
competing designs
Population Ecology
Objectives:
Microbiology
Explore how synthetic biology differs
from genetic engineering.
Molecular Genetics
Investigate and measure the
population growth curve of bacteria.
Enrichment
Employ microbiology techniques.
Properly use synthetic biology and
molecular genetics terms.
BioBuilder Emphasis:
An Engineering Paradigm
Design
Build
Test
The focus of
this
lab
What do we know?
Parts build Devices
E. coli exhibit a predictable growth
curve (lag, log, stat)
The Eau d’coli device designed by the 2006 MIT iGEM team expresses
the banana smell during the stationary phase.
Questions to Consider
1. What happens in the original design?
2. What is the role of the promoter?
3. What is the role of the ATF1 ORF?
4. What is the role of the ATF1 enzyme?
Your Challenge...
We have been sent 4 strains of E. coli. Each has a different composition of
parts. Test each banana generating device to see which will generate the
strongest smell.
Strains
Description
1-1
original device (stat.
phase promoter)
1-2
Stationary phase
promoter with inverter
Strains
Predictions
1-1
smells like bananas
at stationary phase
1-2
smells like bananas
when not in stationary
1-3
log phase promoter
1-3
smells like bananas
when in log phase
1-4
no banana smell
generating device
1-4
no banana smell
PREPARATION
Sequence for Protocol A (Protocol B is an alternative where students can
prepare cultures of each strain and take samples every 20 minutes... this
requires a full day and many classes participating):
1. Prepare cultures of each strain (see prep videos on preparing Liquid
Overnights and Stock Cultures).
2. Place a sample in the refrigerator before it has time to incubate.
Growth Curve
These will be the LAG phase
samples.
3. Let each sample grow for 6 hours on a magnetic stir
plate at room temperature.
These will be the LOG phase
1-1
1-2
1-3
1-4
samples.
4. Let remaining sample run overnight on a magnetic
Time (h)
stir plate at room temperature.
These will be the STATIONARY phase samples.
1-1
1-1
1-2
1-3
1-4
1-2
1-3
1-4
There will be 12 samples all together (4 cultures each at three time points).
PERFORM
TODAY...
For LAG and LOG samples you
will measure:
1. OD 600 using the Spec 20.
This will tell you the population
of bacteria at LAG and LOG
phase. If a Spec is NOT available,
you can prepare samples on the
McFarland Turbidity Scale.
2. The banana smell by
comparison with the banana
standards (prepared from
banana oil) at LAG and LOG
phase.
3. Be sure to replace the cover
on the cultures after smelling.
Coffee in between smells???
Cell Density
1-1
1
1-3
1-2
1:10
dilutions
1-3
1-2
OD600
How many cells?
1-4
McFarland Standards
Smell Test
2
0
1
Mmmmm!
How sweet it
is!
2
3
4
1-1
5
1-2
1-4
1-3
LAG Phase
Banana
Standards
6
SMELL STANDARD
DENSITY STANDARD
SMELL STANDARD
DENSITY STANDARD
1-1
1-2
1-3
1-4
LOG Phase
1-1
1-2
1-3
If you are employing Protocol B, you will need to make 4 data
tables like this, one for each strain
TOMORROW...
1. Repeat for STATIONARY phase samples.
Submit Your Data Here:
http://www.biobuilder.org/activities/
Password: natbioethics