Laura Voss - Synthetic Biology Seminar

Paper Discussion 10/23

Links for "Relative Sensitivities of Various Reporter Genes"

• Ice Nucleation Proteins and inaZ. InaZ is a reporter for ice nucleation proteins, which, according to NCBI, "enable bacteria to nucleate crystallation in supercooled water". Since supercooled water is chilled below its freezing point, ice nucleation proteins form the "seed" around which ice crystals can form in the cell. The more proteins are produced, the more ice crystals will be present in a cell. See the Wikipedia entry on supercooling.

• Sugars used to test promoter's responsiveness to sucrose. Several sugars were tested with the sucrose-responsive promoter to ensure its specificity. Sucrose, fructose, and sorbose were among the sugars tested, and were the only ones that elicited a response from the promoter. Illustrations of their molecular structure are in the next page.

Links for "Simulation of GFP Expression"

• Michaelis-Menten Kinetics is a system of equations that describes the kinetics of enzymes when the concentration of substrate is much greater than the concentration of enzyme. In this paper, Michaelis-Menten kinetics were used to describe the degradation of GFP, or, more accurately, the activity of the protease responsible for the degradation of GFP.

Prior to this paper's publication, the degradation of GFP had been calculated using first-order kinetics. In that case, the degradation of GFP (whether it was fluorescing or not) would have been degraded at a constant rate, and would degrade faster the more protein was present. In theory, the degradation rate could increase indefinitely, so long as the amount of protein continued to increase. In a Michaelis-Menten equation, there is a maximum rate of degradation that an enzyme approaches as the concentration of substrate increases; however, it never reaches this maximum rate. So, after a certain concentration of GFP is reached, the degradation rate remains almost constant no matter how great the concentration of GFP. This can affect how much fluorescence is perceived in a cell and whether it corresponds to promoter activity.

• Lineweaver-Burk Plots are a way of graphically representing Michaelis-Menten kinetic equations. Prior to the introduction of computer software that could easily calculate enzyme parameters, Lineweaver-Burk plots were frequently used to calculate such values as the maximum rate of reaction and the Michaelis constant. However, as the plots are not as accurate as computer calculations, they are now primarily used to represent kinetic data rather than calculate it.