By the conclusion of this laboratory investigation, you should be able to:
- Explain how synthetic biology as an engineering discipline differs from genetic engineering
- Explain the population growth curve of bacteria
- Culture bacteria using proper microbiology methods
- Measure the growth of a bacterial population
- Bacterial Growth Curves
- Define and properly use synthetic biology terms:
- Part, Device, Inverter, iGEM
- Define and properly use molecular genetics terms:
- Promoter Ribosome Binding Site Open Reading Frame Terminator Plasmid
For the 2006 iGEM competition, MIT students designed Eau d’coli, E. coli that smell like bananas when their population is in the stationary phase. They did this by inserting a device that contains a stationary phase sensitive promoter coupled to a banana smell device. The banana smell device contained a ribosome binding site (RBS), an open reading frame (ORF) that encodes the ATF1 enzyme and terminator sequences. The ATF1 enzyme converts isoamyl alcohol to isoamyl acetate, the molecule that gives bananas their characteristic smell.
In this investigation, you’ll try to generate the banana smell during the bacteria’s log phase of population growth. There are two ways (at least!) you could accomplish this. Both approaches use the original banana smell generator device (an RBS, the ATF1 gene and a transcriptional terminator). One approach couples the banana smell generator device to a new part, a log phase promoter. The other approach uses the stationary phase promoter that was used in the original MIT project but adds a genetic inverter between the stationary phase promoter and the banana smell generator device.
These variants have already been made for you and transformed into bacteria. In fact, you have been sent four strains of E. coli to test. Each contains a different device:
- Sample 1-1. The original Eau d’coli device
- Sample 1-2. The original Eau d’coli device but with an inverter added between the promoter and the RBS.
- Sample 1-3. The banana smell generator coupled to the log phase promoter
- Sample 1-4. A strain of E. coli that has no smell generating devices.
Your task will be to grow liquid cultures of these bacteria and measure the intensity of the banana smell as the population moves from lag phase through log phase and into stationary phase. The intensity of the banana smell can be compared to dilutions of banana extract. The population growth can be measured at each of these phases using a spectrophotometer or the McFarland Turbidity Standards.