The Rate of Things

As with the size of things, we use the cell cylce as a temporal ruler to which all kinetic cellular processes can be compared. Students explore the doubling time of typical prokaryotic (E. coli) and eukaryotic (S. cerevisiae) organisms. This also has the benefit of acquainting them with basic spectrophotometric techniques when measuring the bulk doubling time using light scattering.

Below are linear and logarithmic plots of the growth of E. coli and S. cerevisiae, respectively:

The times we recover for doubling are right on target for accepted values: 33 minutes in the case of E. coli and 1:20 hours for S. cerevisiae.

It is now common to measure quantify gene expression by measuring the concentration of fluorescent proteins. In fact, the DNA science part of the Bootcamp and one of the advanced projects are centered on this issue. One of the rates that are useful to keep in mind then is that of photobleaching of the fluorescent protein of interest. We measured the photobleaching of GFP being expressed at high levels in E. coli. The students wrote a Matlab code to measure the fluorescence as a function of time, obtaining a half life of around 20s for the particular setup they used.

Even more interesting than watching a bulk cell culture enter the exponential growth regime is actually imaging individual cells as they divide and grow. In the following movies students used phase contrast, video microscopy to watch S. cerevisiae yeast cells go through roughly 3 cell cycles. We especially like this assay because one gets a feeling for the degree of synchrony in division and fluctuations in division time. The students also gained some practical experience writing image processing code to bring successive images into registry (i.e. remove drift).

Raw microscopy images

After alignment and cropping

The enzyme beta-galactosidase (drawing by David Goodsell)

In a classic assay, students validate the use of linear kinetic theory using the enzymatic activity of beta-galactosidase. In E. coli this enzyme is responsible for breaking lactose down into glucose and galactose, which are two more readily usable forms of energy for the cell. However in our case, we allow the enzyme to work on the lactose analog ONPG. The enzyme breaks this down into galactose and the molecule ONP. ONP having a well characterized absorbance at 420nm is used to track the rate of the enzymatic reaction. The chemical reaction is given by:

Using kinetic spectrophometry they measure the production of ONP as a function of time for varying concentrations oaf the enzyme. This allows them to deduce activity of the enzyme and in some sense validate their understanding of linear kinetic theory. At early times in the reactions the concentration of ONP should be given by:

The students logarithmically sampled the kinetics by varying the beta-gal concentration:

Once the raw kinetic data is collected, the students have an average rate for a given enzyme concentration. This allows to obtain the forward rate of the reaction:

Dictyostelium have an amazing life cycle. They act as indepedent cellular organisms, but under the right conditions they will associate to form fruiting bodies. When a mature fruiting body releases its daughter spores, these spores will forage and eat bacteria in the surrounding area. Below is a picture of a mature fruiting body that has recently 'landed' on the plate and deposited spores.

Once these cells run out of food, they will assess their situation and decide whether to join together and form fruiting bodies. This amazing change of character - from single celled to mutlicellular organism is caught on film below as groups of cells join to form mobile bodies called 'slugs'. These slugs travel and find a spot to begin forming the fruiting body. In this movie two large slugs are visible, with one forming a stalk and fruiting body.

While watching simple single celled organisms live out their cell cycle is an amazing sight, we decided to try and capture something a little more challenging - the zygote formation from raw sea urchin gametes. Students learned how to take live sea urchins and humanely harvest their sperm and eggs. The sperm were diluted and mixed at an appropriate, empirically determined ratio with egg cells.

Sea urchin egg, pre-sperm introduction--------------------After sperm introduction-----------

As with most sexual reproduction, it is necessary that once the egg has been fertilized a mechanism to prevent the entry of more sperm be enacted. The movie below shows the initial stages of separation of the ferilization envelope which is the 'shield' that prevents more sperm from entering the fertilized egg.

Once the fertilization envelope has formed, the zygote will begin its first mitosis. The following movie traces two zygotes through their first 8 divisions (with a minor technical problem mid-movie). A few interesting things to note: in the first division - you can actually see the nucleus split and become pole localized; there are some very precise symmetry rules governing the relative positions of the cells that are followed during the first few divisions; last - there is an amazing degree of synchrony between the zygotes' divisions.