1. The BioBuilder Lab Experience: Eau That Smell! PresentPreparePerform

2. PRESENT The Big Idea: Compare competing designs Objectives: Explore how synthetic biology differs from genetic engineering. Investigate and measure the population growth curve of bacteria. Employ microbiology techniques. Properly use synthetic biology and molecular genetics terms. Where can it fit? Population Ecology Microbiology Enrichment Molecular Genetics

3. BioBuilder Emphasis: An Engineering Paradigm DesignBuild Test The focus of this lab lab

4. The Eau d’coli device designed by the 2006 MIT iGEM team expresses the banana smell during the stationary phase. What do we know? E. coli exhibit a predictable growth curve (lag, log, stat) build DevicesParts

5. 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? Questions to Consider

6. 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. Your Challenge... StrainsPredictions 1-1 1-2 1-3 1-4 StrainsDescription 1-1 original device (stat. phase promoter) 1-2 Stationary phase promoter with inverter 1-3 log phase promoter 1-4 no banana smell generating device smells like bananas at stationary phase smells like bananas when not in stationary smells like bananas when in log phase no banana smell

7. 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).Liquid Overnights 2. Place a sa2. Place a sample in the refrigerator before it has time to incubate. PREPARATION There will be 12 samples all together (4 cultures each at three time points). 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 samples. 4. Let remaining sample run overnight on a magnetic stir plate at room temperature. These will be the STATIONARY phase samples. 1-1 1-2 1-3 1-4 Time (h) Cell Density Growth Curve

8. 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??? PERFORM OD600 How many cells? 1:10 dilutions 1-1 1-2 1-3 1-4 1-1 1-2 1-3 1-4 LAG McFarland Standards LAG PhaseSMELL STANDARDDENSITY STANDARD 1-1 1-2 1-3 1-4 LOG PhaseSMELL STANDARDDENSITY STANDARD 1-1 1-2 1-3 1-4 LOG Smell Test Cell Density 1-1 1-2 1-3 1-4 Banana Standards 0 1 2 3 4 5 6 LAG & LOG 1 2 Mmmmm! How sweet it is!

9. 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.

10. Submit Your Data Here: http://www.biobuilder.org/activities/ Password: natbioethics