Supplementary Information for an Experiment on:
Beta-Galactosidase in Escherichia coli
in the UW-Madison Bacteriology 304 Course

Fall Semester, 2003 and Summer Session, 2007


I.  A General Summary of the Lac Operon.
The movement of RNA Polymerase along the lac operon (i.e., the direction of transcription) is from left to right.

x

  • RNA Polymerase Attachment:  The Catabolic Activator Protein (CAP) attaches to the CAP BINDING SITE (when CAP is enabled by cAMP) and allows RNA Polymerase to attach to the PROMOTER.

  • RNA Polymerase Transcription of the Lac Operon then proceeds – unless blocked by the Repressor Protein (RP) which attaches to the OPERATOR (when RP is not disabled by allolactose).

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.

 

GLUCOSEx
cAMPx


CAP enabled by cAMP to allow attachment of RNA polymerase.

GLUCOSEx
cAMPx


CAP disabled, so RNA polymerase cannot attach.

LACTOSEx
  Allolactosex
*

RP disabled by allolactose, allowing transcription of lac operon to proceed (if RNA polymerase can attach).

RNA polymerase:

  • can attach,

  • and can transcribe lac operon (as RP blockage is disabled).
IDEAL SITUATION

RNA polymerase:

  • cannot attach;

  • therefore, no transcription of the lac operon (even though there is no blockage by RP).

LACTOSEx
Allolactosex


Repressor Protein (RP) is free to block lac operon transcription.

RNA polymerase:

  • can attach,

  • but cannot transcribe (due to blockage by RP).

RNA polymerase:

  • cannot attach;

  • therefore, no transcription of the lac operon (which would be blocked by RP anyway).

*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.)


xA 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.


III.  A Sample Calculation.

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:

x


IV.  Some Data for Graphing Practice.

One may get an impression of the nature of the mutant strain – that is, what mutation(s) may have allowed the results shown?

strain & mediumtime (min)A420A600time of
assay (min)
U/mlU/(ml X A600)
mutant strain in MM+glucose00.0330.038.331.44E-054.80E-04
150.340.057.0331.76E-053.52E-04
300.340.046.671.86E-054.64E-04
450.040.057.02.08E-054.16E-04
600.0660.067.03.43E-055.71E-04
750.1090.127.0335.64E-054.70E-04
900.240.285.331.64E-045.85E-04
1050.2460.245.51.63E-046.78E-04
120
mutant strain in MM+lactose000.00910.000
150.0350.24610.01.27E-055.17E-05
300.0550.30310.02.00E-056.60E-05
450.1980.3768.58.47E-052.25E-04
600.2070.4684.531.66E-043.55E-04
750.2470.5624.332.07E-043.69E-04
900.3490.6082.834.48E-047.38E-04
1050.850.7561.691.83E-032.42E-03
120
wild-type strain in MM+glucose000.035.03.64E-061.21E-04
150.0150.0785.01.09E-051.40E-04
300.0080.085.05.82E-067.27E-05
450.020.175.01.45E-058.56E-05
600.0210.185.01.53E-058.48E-05
750.0240.265.01.75E-056.71E-05
900.0190.45.01.38E-053.45E-05
1050.0160.495.01.16E-052.37E-05
120
wild-type strain in MM+lactose00010.000
1500.0710.000
300.0380.0310.01.38E-054.61E-04
450.0240.0410.08.73E-062.18E-04
600.0780.0610.02.84E-054.73E-04
750.210.0710.07.64E-051.10E-03
900.2470.1410.08.98E-056.42E-04
105
120

Page last modified on 1/10/17 at 1:00 PM, CST.
John Lindquist, Department of Bacteriology
University of Wisconsin – Madison

Damn! This experiment was fun.
Retirement sucks.