Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters

This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing this protein.


Background
Gene dosage experiments are crucial for understanding the effects of protein over-expression on fitness and determining the optimal levels of protein abundance. Several genes are toxic even when expressed at very low levels. It is therefore important to express the protein from a tightly regulated promoter to minimize leaky expression. Here we have elucidated conditions for expression of proteins under the well-characterized arabinose induced pBAD promoter, and designed protocols to measure the intracellular abundance and fitness of E. coli cells harboring the overexpression plasmid.

A.
For abundance measurement

1.
Transform pBAD plasmid containing the gene of interest in to electrocompetent BW27783 cells (see Notes, point #1), and spread on LB/ Agar plates containing 100 µg/ml ampicillin. Incubate plates overnight at 37 °C.

2.
Next day, pick a single colony from the plate and inoculate into 2 ml of supplemented M9 medium (see Recipes and Notes, point #2) containing 100 µg/ml of ampicillin. Grow overnight with shaking (250 rpm) at 37 °C in 14 ml polypropylene tubes.

4.
After 4 h, measure OD 600 of the cultures, and spin down in a table-top centrifuge at 3,000 × g for 15 min. Aspirate as much culture supernatant as possible. Store the cell pellets at −20 °C.

5.
Prepare the lysis buffer, which is the buffer of choice (50 mM sodium phosphate buffer, pH 7.4, 10 mM Tris-HCl pH 8.0, etc.) supplemented with the detergent Bugbuster and Benzonase nuclease (see Recipes below). Based on the measured OD 600 of the cultures, re-suspend the pellets in the prepared lysis buffer such that the final OD 600 is 2.0 (see Notes, point #3). Allow the lysis to proceed for 20 min at room temperature on a rotator-mixer.

6.
Following lysis, spin down the cell debris for 30 min at 7500 × g in a centrifuge that has been pre-chilled at 4 °C.

7.
Separate the supernatant and quantify the total protein in each sample using the BCA assay kit using the manufacturer's instructions.

8.
Load 20 µl of the supernatants on a 12% Bis-Tris SDS polyacrylamide gel, after required dilution of the samples (see Notes, point #4).

9.
Resolve the samples at a voltage gradient of 10 V/cm of gel.

10.
Once the dye front has reached the base, the gel is ready to be transferred to a membrane for blotting. Use pre-assembled nitrocellulose membrane sandwiches (see Notes, point #5).

11.
Following transfer, wash the membrane with water two times to remove transfer buffer components and weakly bound proteins. From this step onwards, use Invitrogen's Western-breeze Chromogenic Immunodetection kit to develop the membrane.

B.
For growth rate measurement

1.
From step A2 above, dilute the overnight culture to a final OD 600 of 0.01 (see Notes, point #3) into fresh M9 medium containing 100 µg/ml of ampicillin and varying concentrations of arabinose (see Recipes). Aliquot 150 µl of this into three wells of the honeycomb Bioscreen plate (see Notes, point #6). This serves as replicates for a single colony.
To obtain standard error of biological replicates, inoculate 3 independent colonies, and subject each of them to varying arabinose concentration.

2.
Aliquot 150 µl M9 medium into three independent wells. This serves as the background for OD measurements.

3.
Measure OD 600 values at 15 min intervals over a period of 12 h in Bioscreen C system at 37 °C with shaking (see Notes, point #6).

2.
Normalize the intensity by the total protein concentration obtained by BCA assay, and also scale up the values by the dilution factor. If the protein of interest is an endogenous E. coli protein, then calculate the fold-overexpression of the protein of interest based on the intensity obtained for untransformed BW27783 cells (set to 1). For a foreign protein, the expression level obtained with 0% arabinose should be set to 1.
conventional microplate reader or spectrophotometer with cuvette can also be used to perform this step successfully.

7.
If it is desirable to find out the absolute amount of protein required to achieve a particular growth inhibition, it can be done by purifying the protein of interest, estimating its concentration and then using a known amount of the purified protein as a standard/reference to estimate the amount of protein from the crude lysate at each given induction level.

1.
Supplemented  A. Plot of relative growth rate as a function of arabinose concentration; B. Plot of relative growth rate as a function of intracellular abundance of DHFR protein.