The Rate of Things:
How fast do cells divide? How do enzyme kinetics depend on concentration?
What is the timescale of early development?
[ Rate of E. Coli Division | Enzyme Kinetics | Development ]
Rate of E. Coli Division
 |
 |
| Optical absorption at 600nm for E. coli in the exponential growth regime - plotted in linear axes. | Optical absorption at 600nm for E. coli in the exponential growth regime - plotted in semi-log form to show the increase is indeed exponential. Simple regression gives a doubling time of 33 minutes. |
| Cells were launched in liquid LB medium, allowed to pass through the lab phase, and then cell densities were measured in a spectrophotometer. One could relate the OD600 to actual concentration using Beer's Law. | |
Enzyme Kinetics - The beta-gal Assay
Beta-galactosidase, one of the classic enzymes used to measure gene expression, is used to digest ONPG, a substract that turns yellow upon digestion. This way the rate of product increase can be easily measured using spectroscopic methods. Our first objective was to determine the range at which the reaction is in the linear regime. That is: what is the ranges of beta-gal concentrations where concentration and the rate of digestion a related linearly?
This experiment serves not only as a great example of "The Rate of Things", it is also useful to introduce the students into basic concepts of protein handling in the lab, the required buffers and the used of a spectrophotometer.
For each beta-gal concentration the amount of product as a function of time is obtained and the initial rate of digestion determined.
|
The obtained rates as a function of enzyme concentration are plotted and the linear range of the reaction is determined. |
Even though we are now able to determine where the linear range is a lot of questions still remain open. Future work will include determining the concentration of enzyme through spectroscopic methods in order to campare it to the one obtained through this kinetic method.
Sea Urchin Fertilization and Early Development
 |
 |
| Real time video of the fertlization of sea urchin eggs, notice the shedding of the outer cellular envelope after a sperm has entered. | Time-lapsed video, sped up ~900 times showing the first few divisions of the sea urchin embryo |
| In both cases, gametes were extracted from living sea urchins, but kept separate until fertilzation. In the second video, fresh sea water had to be added to keep the solution relatively isotonic with the developing embryo; this is the disturbance one sees during the video. | |