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Learning Module - Experiment 9B

Assay of Crude Lysates for Topoisomerase I Activity

Construct a table to enter your activity data estimated from the gel. Your 1:1000 dilution of pure Topo I contains 4 ng protein / μL (yours may be a different concentration).

What is loaded in each lane.

Then begin to interpret your results:

Different tertiary structure migrate at different rates.

Topoisomerase I activity is observed as a distribution of bands due to the stochastic (random) nature of the enzyme reaction. Just as growing bacteria do not normally divide in unison, Topoisomerase I does not act on all of the pUC12 molecules in unison. As a result, some pUC12 molecules have more supertwists removed than others, and a distribution of bands representing different topoisomers of the plasmid are observed. The activity for each sample is taken as the average change in linking number, or the midpoint of the distribution of bands (rungs). The midpoint need not be an integer; if two adjacent bands display equal intensity, the activity is taken as being midway between them.

Fill in the appropriate entries on your table:
One Unit of enzyme is the amount of enzyme that will reduce the linking number of 1 μg of pUC12 plasmid to 1/2 the original linking number, in 30 min at 37 C

In this example case, pUC12 has a linking number of -7, thus 1 Unit = 3.5 rungs (your results may show more rungs than this example)

Add results to table

Construct a graph of Activity vs. ng of TopoI:

Activity units vs nanograms of protein.

The slope of the line where the data are linear yields Specific Activity. The datum at 20 ng TopoI has not been included because it does not follow the linear trend of the other three points.

Determine the protein concentration of your +IPTG and -IPTG lysates:

Construct a standard curve for the estimation of the protein concentration in the lysates

BSA standard curve

Estimate the protein concentration in each of your lysates (for this module we estimate 10 mg protein / mL for the +IPTG lysate and 8 mg protein / mL for the -IPTG lysate) and enter these data into your table after accounting for the dilutions made to each lysate, in this case 10-fold for -IPTG and 100 fold for +IPTG:

Adding standard curve data to the data table

Repeat the analysis for your +IPTG and -IPTG extracts:

Activity estimates

Enter the data into your table,

Unit calculations for +IPTG

Unit calculations for -IPTG

And construct a plot for each lysate:

Slope for +IPTG

slope for -IPTG

Finally, compare the +IPTG and -IPTG specific activities and estimate the -fold overproduction:

Calculation of fold increase

and the percent of protein in the +IPTG extract that is Topoisomerase I:

Calculation of percent TopoI