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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