Stressing the Lac-Operon

www . Science-Projects . com

Lac-Operon Stress Test
Classtime: 45-60 min

    EQUIPMENT AND SUPPLIES: E. coli K-12 or similarly inducible lac+ strain, nutrient agar (without any sugars), lactose, spectrophotometer (420 nm and 540 nm), cuvettes, test tubes, stresser reagents or conditions, air-incubator (43°c), vortex mixer, clock with second hand, ONPG, two 100μL pipetters with yellow tips

    REVIEW: Expression of the lac-operon in E. coli requires the presence of lactose with no other sugar present. During the process of induction the starving cell's dwindling supply of ATP allows for the derepression of adenyl-cyclase for the synthesis of cyclic-AMP (cAMP), which is required by transcriptase for the reading of the lac-operon, which has simultaneously been derepressed by the inactivating activity of lactose on lac-repressor protein. If other sugars are present, those are preferentially sent to glycolysis and ATP levels are increased shutting down cAMP synthesis and thus lac-operon transcription. In normal lactose utilization, products are glucose and galactose (gal is converted to glucose by the expression of yet another operon). Since these sugars become present, the ATP pool is raised with consequent depression or damping down of the lac-operon expression. Thus under normal conditions, the lac-operon is only partially "on."

    It is our plan to stress the cells in various ways such that the ATP pool might be drained so that lac-expression is increasesd to levels above what otherwise would be normal.

    Earlier students reasoned that because active transport has a strong requirement for ATP, subjecting the cells to osmotic stress should deplete ATP, and thus - if our model is correct - cause hyper-expression of the lac-operon such that elevated levels of lactase (aka β-galactosidase) could be measured using the methods of enzyme kinetics as that lactase hydrolyzes ONPG (ortho-nitro-phenyl-β-D-galactopyranoside) to yield a yellow color that increases the longer the reaction continues.

    Let's set up two experimental runs that should be stressful to the E. coli. One will be osmotic stress, and the other will be thermal stress, and both will be performed initially in petri plates rather than in liquid culture.

Osmotic StressThermal Stress
  1. Make a series of nutrient agar + 0.5% lactose plates such that their sucrose concentrations range from 0% to 15% (0, 1, 2, 4, 6, 10, 15%). Don't forget to label the BOTTOMS of the plates! (Since we don't know the susceptibility of E. coli to non-digestible sucrose, we use this semi-logarithmic series such that if they are extremely sensitive, the upper plates should not grow, and if, on the other hand, they are relatively insensitive, then we shall make use mostly of the higher concentrations plus the 0% (control).)
  2. Inoculate the plates by swabbing lawns of E. coli completely over their surfaces.
  3. Allow to incubate at room temperature. (Remember, we haven't yet tested the temperature effects.) The cultures will be ready the next day.
  1. Make two nutrient agar + 0.5% lactose plates. Label the BOTTOM of one RT for room temperature, and the other's bottom 43°c.
  2. Inoculate the plates by swabbing lawns of E. coli completely over their surfaces.
  3. Allow the RT plate to incubate at room temperature, and the other plate in the 43&@177c incubator. The cultures will be ready the next day.
  1. As the cells are in stationary phase by this time, you can do things with them without their changing. Add about 10 mL tap water to the surface of each plate. Using a swab, slurry up the bacteria. Get as much of the bacteria free of the agar as possible without being a "farmer" "plowing" the agar. We are testing the bacteria, remember? And not the agar.
  2. Pour the slurries into properly labeled test tubes.
  3. You now want to adjust the slurries so that they are close to having an absorbance (540 nm) of 1.0, and you will do this by first measuring the abs or each tube, then adding some water, swirl to mix, re-read its abs, and so on until you get a reading in the range of 0.9 to 1.1. (RECORD your reading! It will be called your STARTING VALUE.)
  4. To disrupt and permeablize the cells, to each 5 mL of cell suspension, add 2 drops of chloroform (CHCl3) and 2 drops of 0.1% SDS (sodium dodecylsulfate; think "SuDS" - it's a detergent!). Note the droplet of water-immiscible CHCl3 lying at the bottom of the tube.
  5. Vortex the tubes violently for 15 seconds ("1 elephant, 2 elephants...") (This homgenizes the chloroform and with the action of the SDS, the membranes are dissolved leaving the bacteria as mere cell-wall-mesh sacks full of big molecules but meshy enough for small molecules such as ONPG and its yellow product to get in and out of the sacks.)
  6. Readjust your spectrophotometer to 420 nm (green) so that it can read yellow.
  7. Insert a permeablized cell suspension such that the cuvette is about 80% full.
  8. Zero the machine. (No yellow yet!)
  9. You need three people now:
    • Meter reader
    • Clock reader
    • Secretary who has a partially made table with two columns. Under time: "0, 10, 20, 30, 40, 50, 60."
  10. The clock reader and the secretary both load up their 100μL pipetters with ONPG solution.
  11. As the clock's second hand is approaching 12, the meter reader says "Ready!" Both pipetters have their tips at the opening of the cuvette that the meter reader is holding.
  12. When the second hand reaches 12, the meter reader says NOW!, and both pipetters are squirted into the cuvette.
  13. IMMEDIATELY the meter reader puts a finger over the mouth of the cuvette and inverts it a couple of time to mix, and rushes to insert the cuvette into the machine. Meanwhile, the clock reader watches the second hand click onwards, and the secretary assumes note-taking position.
  14. NOW! says the clock water as the second hand hits "2", and that triggers the meter reader to recite the meter's reading, which the secretary writes down.
  15. And so this goes on as the second hand reaches each 10 second mark on the clock.
  16. After the 60-second reading, the cuvette is lifted out so that all can now see that it is yellow. The cuvette is then cleaned out, and another group can use the spectrophotometer for their cell suspension.
  17. Back at their desk, the students divide each of their readings by their "STARTING VALUE" (see step #2, above).
  18. Graph those values on bi-linear graph paper.
  19. Add the curves obtained by the other groups.
  20. Discuss any trends.
  21. Discuss the value of using chemical analogs (ONPG is an analog of lactose), and their place in medicine and pharmaceuticals, cancer treatment, pesticides, and in the mere periodic table of the elements where S is an analog of O, etc.