Supplementary Information for an Experiment on:
I. A General Summary of the Lac Operon.
II. A Review of Activation and Repression of the Lac Operon
based on factors which allow (or block) attachment of – and transcription by – RNA polymerase.
*Basal enzymatic activity is always present which allows the initial production of allolactose when lactose is introduced into the system. (Even though no beta-galactosidase production is expected when glucose is present, there is still a low level of transcription.)
A question from a Bacteriology 304 quiz given August, 2007: What should theoretically happen when a typical strain of Escherichia coli is inoculated into a medium containing high amounts of both glucose and lactose? The two-part answer:
Application of the Boolean circuit idea to the lac operon is discussed here.
A flow chart for a timed beta-galactosidase activity experiment is shown here. The "assigned culture" may be a wild-type or mutant strain in a standard broth medium containing glucose, lactose or both. (Why not see what happens in a galactose broth.)
The following is an example of a calculation to determine units of beta-galactosidase per ml of a normalized culture in a timed experiment where we are testing (with a spectrophotometer set at 420nm) a 4 ml assay tube composed of 1 ml of culture, 1 ml of buffer, 1 drop of toluene (an insignificant amt.), 1 ml of ONPG, and 1 ml of stop solution.
Comparison is made with a cell absorbance reading (determined at 600nm) of the same culture at the same time of testing for the normalization of the result (i.e., based on constant cell density).
For this example calculation we have the following data that are plugged into the sequential formulas:
One may get an impression of the nature of the mutant strain – that is, what mutation(s) may have allowed the results shown?
|strain & medium||time (min)||A420||A600||time of|
|U/ml||U/(ml X A600)|
|mutant strain in MM+glucose||0||0.033||0.03||8.33||1.44E-05||4.80E-04|
|mutant strain in MM+lactose||0||0||0.009||10.0||0||0|
|wild-type strain in MM+glucose||0||0||0.03||5.0||3.64E-06||1.21E-04|
|wild-type strain in MM+lactose||0||0||0||10.0||0||0|
Damn! This experiment was fun.