Science Buddies: "Ask an Expert"

Hello. I was wondering if someone could please help me sort out some of the details on my science fair project for this year. I really want to study the ways different stimuli, like temperature variation or different concentrations ofmedia and how they affect the regulation of a single E. coli gene. I was wondering if I would have to order a plasmid that incorporates an E. coli gene and if I would be able to successfully determine how different stimuli affect the regulation of a gene. I was also wondering if I could measure that gene through PCR and gel electrophoresis and would help me show if the gene was properly regulated, or turned on and off at the correct times.

Hi Nik,

Gene regulation in higher organisms is a vast and complex subject, so working with E. coli is a good idea. Studying bacteria was how scientists learned the basics of gene regulation in all organisms. Many bacteria are exposed to a constantly changing environment and have had to adapt by evolving a variety of regulatory systems to turn on or off specific genes, usually those involved in metabolism.

Genes code for proteins, some of which are enzymes and it is these biological catalysts that E coli use to metabolize certain chemicals such as sugars or carbohydrates. Some enzymes are always present meaning that their genes are being transcribed and translated into proteins all the time. Others are known as inducible and those are the ones you are interested in because they are influenced by temperature, pH, nutrients and other environmental factors.

One of the main jobs of E coli is to make the most of its food supply and these bacteria need some kind of sugar to use as a carbon source. The default is glucose and most of the enzymes for absorbing and digesting this sugar are always present. But when other sugars such as sucrose, lactose, fructose, mannose or galactose are present instead of glucose, E coli has to switch gears. That’s where gene regulation comes in.

For example, when galactose is available a signal is sent to the galactose ‘regulon’, the name of the gene complex responsible for galactose metabolism. The gene for making galactose transporter is turned on and galactose is transported inside the bacteria. Other genes are then turned on that break down the sugar. Here’s a paper about this: http://www.ncbi.nlm.nih.gov/pubmed/7934815

I would suggest using sugar metabolism in E coli K12 as a regulatory system because it is relatively easy to manipulate the growing conditions for the bacteria by giving them different sugars and the result can be measured in terms of growth and increase in cell numbers. You could get several different kinds of sugar and add them separately or in combination to E coli in liquid cultures, allow the bacteria to grow for a few hours then do serial dilutions and plate counts. You can look up the published gene regulation information and relate it to the differences in bacterial growth rate that you observe. If you have access to a PCR instrument and agarose gel electrophoresis unit then you could measure specific changes in a transcription factor, repressor or gene product.

In addition to the links Lisa gave you about E coli, you should read the general information on working safely with bacteria:
https://www.sciencebuddies.org/science- ... fety.shtml
https://www.sciencebuddies.org/science- ... #procedure

Youtube has many videos about how to count bacteria and here’s a good one: http://www.youtube.com/watch?v=pmRUBYlPMBM

Let us know when you have more questions and we will help you with the details.

Good luck!

Sybee

Thank You so much! This sounds really interesting!
I was wondering if I could do something similar with C. elegans because a few of the studies I have looked at, have shown that C. elegans have been a great model organism.

Hi,

Yes, you could use C. elegans as your test organism, but the experiments would be complicated by the fact that the worms are multicellular and have organ systems and reproductive cycles. They are also more difficult to grow and test.On the other hand they are a great model organism for many types of studies from longevity (http://www.google.com/url?sa=t&rct=j&q= ... 7519,d.eXY) to Alzheimer's disease (http://www.google.com/url?sa=t&rct=j&q= ... 7519,d.eXY).

If you want to make a project featuring C. elegans, let us know and we will help you with the details.

Sybee