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 engineering paradigm and the role of tuning a system.
- Explain the functioning of the lac operon and relate it to this lab.
- Culture bacteria using proper microbiology methods.
- Measure a kinetic chemical reaction:
- Define and properly use synthetic biology terms:
- Part Device Inverter
- Define and properly use molecular genetics terms:
- Promoter Ribosome Binding Site Open Reading Frame Terminator Plasmid
Some Biodesign Principles
As engineers, synthetic biologists engage in the design–> build–> test cycle. They designgenetic parts, devices and systems. For example a bacterial gene expression device might be designed by thoughtfully coupling together a promoter, a ribosome binding site (RBS), an open reading frame (ORF), and a terminator sequence. Synthetic biologists can also build the devices they design, using techniques such as DNA synthesis, gel electrophoresis, polymerase chain reaction, and cloning. To close the loop on the design/build/test cycle, it’s important for synthetic biologists to have good ways to test the function of the cells they’ve built. This might mean characterizing the cells behavior with enzyme activity assays, fluorescent protein measurements or phenotype analysis. Depending on what the system is suppose to do, measurements might be made to test the speed of a device’s response, its sensitivity to environmental signals, or its level of a protein output.
It’s tempting to think that a strong quick response is always going to be the “best” when designing genetic systems, devices or parts. However, depending on the particulars of the design specification, the output might need to be held at intermediate levels, or even to slow and low outputs in some cases.
There are a few places in the DNA –> RNA –> protein pathway that let us “tune” the output. It’s possible to control the rate of transcription initiation, choosing a promoter that’s active only under some conditions, for example. Translation control is another way to control the output, increasing or decreasing the translation initiation rate by modifying the sequence of the ribosome binding site for instance.
Finer tuning can be achieved by rational combination of promoter and RBS elements, but it’s not trivial to predictably design this way. Some devices end up dependent on others in the cell (they are not fully insulated) and other devices demand a lot of the cell’s resources, slowing a cell’s growth rate if they really demand a lot. Imagine a car in which the volume button on the radio also turned the steering wheel, or a car in which the louder you played the radio, the slower the car could run. This is the current state we face when we engineer genetic systems, devices and parts…problematic to say the least!
About your experiment
Understanding the performance of a device, even a rationally designed one, is needed to reliably engineer genetic systems. Your measurements will compare a designed device to a reference one made from a strong log phase promoter, a strong RBS, a lacZ ORF that produces beta-galactosidase. The designed variants in your experiment all contain the same lacZ ORF, but the devices vary in the efficiency (“strength”) of the promoters and RBSs. You will measure the output of each device, presuming that the combination of promoter and RBS can explain any differences detected in measured beta-galactosidase activity.
The lacZ ORF can be used to measure the activity level of each promoter/RBS combination since the beta-galactosidase enzyme that is encoded by the lacZ ORF allows the bacteria to metabolize lactose (look up information about the lac operon if you need a refresher about this). Normally lactose is cleaved into two monosaccharides, galactose and glucose. You will provide the cells with ONPG (o-nitrophenyl-β-D-galactoside) rather than lactose. When the ONPG reacts with the beta-galactosidase enzyme, the ONPG gets converted into galactose and o-nitrophenol, a yellow compound. Because the intensity of the yellow color is proportional to the amount of beta-galactosidase enzyme in the cells, the intensity of the yellow color measures the output of the promoter/RBS/lacZ ORF combination. You can measure the intensity of the yellow color using a spectrophotometer, like a Spec 20, or with visual comparisons to yellow paint-chip standards.