As was already suggested in the DNA Science section, the issue of choosing a reporter to measure the level of gene expression and characterization of genetic circuits is not trivial. If what we are measuring is related to the processes of regulation before translation the information we obtain from any experiment should be independent of the particular reporter we are using.

To address this issue we measured the activity of the GFP genes in three different ways: at the bulk, single cell and mRNA level. All of these were done on E. coli carrying a tetracycline inducible plasmid controlling the GFP gene (pZE21-GFP). As a result all three measurments can be plotted simultaneously.

Future editions of this project will also include measuring the activity of a reporter enzyme, beta-galactosidase (LacZ), in bulk.

 

Bulk GFP Measurement

Cells containing a plasmid with GFP under the control of a tetracycline inducible promoter were grown at different concentrations of inducer (ATC) and their OD600 and GFP fluorescence monitored as a function of time. The plate reader allowed us to shake the cell cultures between reads and control the temperature, thus the cells were grown directly in the reader. This type of measurement gives us not only the end-point (steady state) fluorescence, it also gives us information about the kinetics of induction.

 

Single Cell GFP Measurement

The fluorescense of E. coli the as a function of the concentration of tetracycline. The approach was to take phase contrast and fluorescence images of the same field of view. The students wrote custom Matlab which allowed them to recognize the cells in the phase contrast image and then integrate the GFP fluorescence over the area of each cell.

Phase Contrast

Fluorescence

Individual cells recognized by our Matlab code

Overlay of fluorescence with the recognized cells

With this tool we can now obtain single cell statistics such as the average fluorescence per cell as a function of the concentration of inducer (ATC).

 

Measuring the GFP mRNA concentration

Using GFP mRNA specific probes we performed a Taqman assay of cells grown in different concentrations of inducer, giving a fluorescent signal proportional to the amount of amplified DNA. Depending on the average concentration of GFP mRNA per cell in each culture there will be a different cycle number at which the signal becomes detectable. In this fashion relative levels of mRNA can be obtained.

Preliminary comparison between the three measurements

From the previous graph it can be seen that, even though the induction curves seem to follow a similar trend, the relative changes in gene-expression are different. This is nowhere near conclusive, but it is a good start especially taking into account that these are the first experiments regarding gene expression performed by most of the students and that these three different assays were all done in only two days.