20.109(F09): Mod 2 Day 1 Testing an engineered biological system

Testing an engineered biological system

Introduction

Why engineer biological systems

Two component signaling (2CS) systems

Bacteria as pixels

E. coli do not normally respond to light but a recent publication describes a combination of genes that lead to light-responsive expression of beta-galactosidase in E. coli. When the cells are grown in the dark, transcription of lacZ is high and the indicator in the media turns black. When the cells are grown in the light, there is very little transcription of the lacZ gene and the media’s natural color (yellowish) shows through.

Bacterial Signaling for Photography System

"coli-roid" example

B-gal as readout

For today, you can get used to this system with a few simple tests. You’ll plate a layer of cells on indicator medium in two dishes, and then incubate one dish in the light and the other in the dark until next time. You expect the dark grown plate to turn a dark color and the light grown plate to stay light. You’ll also grow some liquid cultures under the same light and dark conditions to measure beta-galactosidase activity next time. Finally, you'll familiarize yourself with the assay for beta-galactosidase activity by measuring the activity found a bacterial strain that overproduces the enzyme.

Protocols

Part 1: Test in solid media

Some media has been prewarming in the 42°C incubator for you. The media is traditional LB supplemented with ferric ammonium citrate and “S-gal,” an artificial substrate for beta-galactosidase. When the enzyme cleaves S-gal in the presence of iron, a black precipitate forms changing the color of the media. The media also has agarose so it will harden as it cools. All manipulations should be with your best sterile technique to avoid contamination of your cultures.

1. Add antibiotics (50 μl of ampicillin and 50 μl of chloramphenicol) to the prepared media. These will maintain the plasmids in the bacteria as the cells grow. Use the stir plate to mix but do not allow the media to remain at room temperature for long since it will begin to harden as it cools.
2. Add 200 μl of cells to the media. Stir as before.
3. Pour the volume into two Petri dishes (approximately 20-25 ml in each) and allow the media to harden on the bench before moving the plates into the 37° incubator that is now equiped with a red-filtered lamp. One Petri dish should be placed in a box as the “dark” sample and the other can be placed into the incubator covered with Saran wrap (no lid). Use a pair of scissors to cut several slits in the Saran so moisture can escape.
4. Expose the dishes to red filtered light (0.08-0.15W/m2 650nm range, for 24-48 hours.

Part 2: Test in liquid media

1. In a 15 ml falcon tube, pipet 10 ml of LB
2. Add 10 μl of ampicillin and 10 μl of chloramphenicol.
3. Add 10 μl of your cells. Invert several times to mix.
4. Place 5 ml into snap-cap tubes.
5. Add one dish to your “dark” box and place the other in the incubator on the nutator under the red light.

Part 3: Beta-galactosidase assay

With this assay you will determine the amount of beta-galactosidase activity associated with a sample of bacterial cells. These cells were engineered to overproduce beta-galactosidase. You will dilute the cells and measure each dilution in duplicate to gain some confidence in the values you measure. A table is included here to help you organize your assay, but you can make one of your own if you prefer.

Tube #	Sample	OD600	Time started	Time stopped	Time elapsed (calculated)	OD420	OD550	Units (calculated)
0	blank		0:00
1	undiluted cells		0:10
2	undiluted cells		0:20
3	1:10 dilution		0:30
4	1:10 dilution		0:40
5	1:100 dilution		0:50
6	1:100 dilution		1:00

1. Retrieve 3 ml of the bacterial sample you'll be measuring, called "NB5."
2. Make 1 ml of a 1:10 dilution of NB5, using Zbuffer as the diluent, then use 100 ul of this dilution to make another 1:10, for a final concentration that's 1:100.
3. Transfer 650 μl of the 1:10 dilution to a cuvette and measure the OD600 of this sample. Blank the spectrophotometer with Zbuffer or water. The density of the 1:10 dilution should be in the linear range of the spectrophotometer and your reading can be used to calculate the OD600 of your undiluted cells and your 1:100 dilution.
4. Add 400 μl of Zbuffer to 7 eppendorf tubes labeled 0-6.
5. Add 100 μl of the appropriate cell dilution to each tube. See chart above for guidance. Add 100 ul of Zbuffer to tube 0, to serve as your blank.
6. Next you will lyse the cells by add 20 μl of 0.1% SDS to each eppendorf.
7. To better lyse the cells, you should also add 30 μl of chloroform (CHCl₃) to each tube. Do this in the hood since chloroform is volatile and toxic. You will need to hold the pipet tip close to the eppendorf as you move between the chloroform stock bottle and your eppendorfs since chloroform has a low surface tension and will drip from you pipetmen. Be sure to dispose of your pipet tips in the chloroform waste container located on the right side of the hood.
8. To really really lyse the cells, vortex the tubes for 10 seconds each. You should time these precisely since you want the replicates to be treated as identically as possible.
9. Start the reactions by adding 100 μl of ONPG to each tube at 10 second intervals, including your blank.
10. Stop the reactions by adding 250 μl of Na₂CO₃ to each tube at 10 second intervals once sufficient yellow color has developed. “Sufficient” is defined as yellow enough to give a reliable reading in the spectrophotometer, best between 0.3 and 1.0. Use today's practice assay to learn what this looks like. Be sure to note the time you are stopping the reactions. Also be sure to remember that adding the Na₂CO₃ makes the reactions more yellow.
11. When all your samples have been stopped, add 250 μl of Na₂CO₃ to the blank and spin all the tubes in the microfuge for 1 minute at 13,000 RPM to pellet any cell debris.
12. Move 0.7 ml of each reaction to plastic cuvettes. Avoid the chloroform that will be at the bottom of your tubes. If you add this to the cuvettes, it will "etch" the windows and mess up your readings.
13. Read the absorbance at 420nm. These values reflect the amount of yellow color in each tube.
14. Read the absorbance of each at 550 nm. These values reflect the amount of cell debris and differences in the plastic cuvettes themselves.
15. Dispose of your samples properly: liquid contents of cuvettes can go down the sink, empty cuvettes can go in the sharps containers, eppendorfs with CHCl₃ can go into a waste bottle in the hood.
16. The β-gal activity in each sample is reported as "Miller Units" according to the following formula:

1 Miller Unit = [math]\displaystyle{ 1000 * \frac{(Abs{420} - (1.75*Abs{550}))}{(t * v * Abs{600})} }[/math]

where:
Abs 420 is the Spec 20 absorbance at 420 nm. It is a measure of the yellow color produced by the β-gal activity.
Abs 550 is the Spec 20 absorbance at 550 nm. It is a measure of cell debris.
Abs 600 is the Spec 20 absorbance at 600 nm. It is a measure of the cell density.
t is the reaction time in minutes.
v is the culture volume in mls.
You can average the duplicate values for each sample unless you know of a reason not to (e.g. one tube spilled or had the incorrect amount of something added to it...)

DONE!

For Next Time

Calculate the units associated with the b-gal assays you ran. Are the values for all three of the dilutions identical? If so, why? If not, why not? In other words, please describe some of the variables that affect the confidence you have in the data.

Reagents

• Strain NB334
  • LB supplemented with Sgal
• Strain NB5
  • Z-buffer
  • ONPG
  • Na2CO3