CHEN 4800/5800: Bioprocess Engineering, Spring 1996
University of Colorado
, Chemical Engineering Dept.
Dhinakar Kompala

Lab Exercise 3: Recombinant Bacteria Growth and Foreign Protein Production

Objectives

In this laboratory exercise, you will characterize the kinetics of recombinant bacterial growth and foreign protein expression in shake-flask cultures as well as computer-controlled bioreactors. The goals of the experiment are to determine whether the specific growth rate of the recombinant bacteria is affected similarly by the increased foreign protein expression due to induction in the small scale shake-flask cultures as well as the larger scale controlled bioreactors, and by the plasmid replication alone compared to the plasmid-free host cells in uninduced shake flask cultures.

Background

In this experiment, you will be using two different types of E. coli - one is a plasmid-free host strain designated D1210, and the other is this same host transformed with plasmid pBC26. This plasmid contains the lac Z gene coding for -galactosidase under the control of the tac promoter, which can be induced by IPTG (isopropyl-1-thio--D galactopyranoside). IPTG will bind to the lac repressor, thus releasing the promoter from its repression. The plasmid also contains a gene coding for resistance to ampicillin. A map of the plasmid is shown below.

Upon induction, much of the cells' metabolism is directed toward producing the foreign protein, and the specific growth rate will therefore decrease. You will measure the growth rates of different cultures of E. coli to indirectly determine this metabolic burden upon the cells.

You will also measure the amount of -galactosidase produced from each of the E. coli cultures by performing a chemical assay for this enzyme. You will take a sample from your culture, sonicate it in order to liberate the -galactosidase from the cell, and then add an extract from this sonicate to a solution containg a substrate for the enzyme. This substrate is ONPG (o-nitrophenyl--D-galactoside). The -galactosidase will cleave the ONPG into two products, o-nitrophenyl and D-galactose. The o-nitrophenyl has a yellow color, and the intensity of this color determined spectrophotometrically at 420 nm is proportional to the product concentration in the sample. The rate of product formation, given by the slope of the optical density with time, is proportional to the -galactosidase concentration in the sample.

Experimental Conditions

The experiment will be conducted in three shake flasks cultures inoculated with the following E. coli cells:

Flask 1: Plasmid-free cells (D1210) without inducer

Flask 2: Plasmid-bearing cells (pBC26/D1210) without inducer

Flask 3: Plasmid-bearing cells (pBC26/D1210) with 50 µg/ml IPTG added two hours ahead.

The last two shake flask culture experiments will be duplicated in two slightly larger scale (1 liter) computer controlled bioreactors to determine if the results are duplicated in scale-up.

The cells will be grown in a defined medium containing salts, amino acids, a vitamin, and glucose. The plasmid-bearing cultures will be grown with ampicillin at a concentration of 50 µg/ml in order to select for the plasmid-containing bacteria by killing the bacteria which have lost the plasmid and therefore have lost their resistance to ampicillin. We will maintain the culture temperature at 37°C. The cultures will be inoculated 2 hours before you take your first sample. Samples will be taken every half hour for a total duration of six hours.

Laboratory Procedures

1) Cell Concentration Determination

a) Zeroing the spectrophotometer. Set the wavelength to 600 nm. Using the knob on the left, set the reading to 0% transmission when the chamber is empty; using the knob on the right, set the reading to 100% transmission when the chamber contains a test tube with about 4 ml of deionized water.

b) Determining cell concentration. Remove your group's flask from the water bath. Gently shake the flask to ensure that you get a representative sample from the flask. Flush out the sample tubing by twice pulling out and then pushing in the plunger on the syringe. Again gently shake the flask. Next, fill the syringe with 3-4 ml of sample, unscrew the syringe, and quickly transfer this sample to a glass tube. Quickly screw the syringe back on to the needle. Clean the outside of the test tube with ethanol, insert it into the spec, and take the absorbance reading. Dilute a portion of your sample with deionized water if the reading is greater than 0.250 in order to ensure that your absorbance reading is proportional to the bacterial cell concentration. Record the time you take the sample along with the absorbance reading on a sheet of paper for your own records and on a separate sheet placed in the lab for everybody's records.

2) -galactosidase Assay

a) Take 1.0 ml of your undiluted sample and transfer it to a plastic eppendorf tube. Label the tube with the flask number and sample time. Centrifuge the tube for 5 minutes in an eppendorf centrifuge at 14,000 rpm. Pipet off and discard the supernatant.

b) Resuspend the pellet in 1.0 ml of Phospate Buffered Saline (PBS). Put the sample on ice and sonicate it for 60 seconds with the sonicator output setting at 2, the duty cycle set at 50%, and the pulses set for one second each. Always wear protective covering for your ears when using a sonicator!

c) After sonication, again spin the tube in the centrifuge for 5 minutes at 14,000 rpm. Save the supernatant!

d) Add 1.3 ml Assay Buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1.0 mM MgSO4, 5 mM dithiothreitol, pH adjusted to 7.0; buffer already prepared) to a cuvette. Cover the cuvette with parafilm.

e) Add 50 µl of sonicated supernatant (from "c" above) to the cuvette.

f) Add 0.2 ml of a solution of 4 mg/ml ONPG (o-nitrophenyl--D-galactoside) to each cuvette; pipet up and down several times to mix.

g) Running the spectrophotometer. Press "Prog" and Program 0: HSHI will appear. Press "R/S". You do want to calibrate, so next press "0" and then "Enter". Insert the "blank" cuvette into the cuvette chamber in position 1 and press "R/S". After a short time for calibration, the spec will "ask" you to insert the samples and then press "R/S" to read.

h) Place the cuvettes (yours and the other two groups' will run in the spec at the same time) in the sample chamber of the temperature-controlled Beckman DU-50 spectrophotometer (temperature will be controlled at 37°C) in positions 2-4. Leave the "blank" cuvette in position 1.

i) Record absorbance at 420 nm over a period of 8 minutes by pressing the "R/S" button.

j) The b-galactosidase activity is calculated from the rate of change of the optical density for each sample and is expressed as U/ml (1 unit (U) hydrolyzes 1 µmole ONPG to o-nitrophenol and D-galactose per min at pH 7.3 and 37°C) by comparing the slope (rate of change of absorbance) for the sample to the slope for a -galactosidase standard. The spec is programmed to calculate the slope of your sample; you need to press a few buttons before you will get this data.

1. After your samples have run for 8 minutes, the spec will "ask" for a cell number. Press "2" and "Enter".

2. The display will read "blank"; press "Enter" for the default setting.

3. The display will read "Time 1"; press "3" and then press the "Enter" button. We will only take the data from 3 minutes to 8 minutes for our assay; the first 3 minutes are to allow the cuvettes to warm up to 37°C in the sample chamber.

4. The display will read "Time 2"; press "Enter" for the default setting (8 minutes).

5. The display will read "Factor"; press "Enter" for the default setting.

In a few moments the data will appear at the bottom of your printout. Repeat steps 1-5 for cell numbers 3-5. After this is done press "0" and "Enter" in order to exit the program. Remove your 5 sample cuvettes and the blank from the spec.

The last column of data for each cell (chamber) is "Result"; this is the slope of the absorbance readings for your sample from 3 to 8 minutes. You can determine the U/ml for your sample by comparing your slope to a -galactosidase standard. The slope of a 1 U/ml standard under these assay conditions is 0.0487. A more exact expression of the enzyme activity is U/cell number. You can determine this quantity for your samples; an absorbance of 0.500 at 600 nm reflects a bacterial cell concentration of approximately 5.0 x 108 cells/ml, and this relationship is linear down to the lowest absorbance values with which we are working.

Report (due April 9, 1996)

Using the collected data from all the groups, draw semilog graphs of ln absorbance vs. time for the three shake-flask cultures and the two bioreactor cultures, and the regular graph of -galactosidase content (U/106 cells) vs. time for all the five cultures. Determine the maximum specific growth rate for each of the cultures. Discuss the results, focusing on any differences in µmax and any correlation there may be between cell growth rate, IPTG induction, and -galactosidase production.


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Prof. Dhinakar S. Kompala / Department of Chemical Engineering
College of Engineering / University of Colorado