Controls of Lactase Activity

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Gene Induction Control of Lactase Activity: Genetic and Enzymatic Levels of Control Enzyme Repression

This is a set of inexpensive protocols for use by teachers in their classroom labs. The methods are divided into two levels of difficulty: one is qualitative and is quicker, less complicated and touches the high points, while the other is quantitative and more time-consuming because it involves measuring the rates of induction and of repression. In either level, it is strongly suggested that the teacher who is a first-timer at this lab maintain close contact with the author of this website, or with someone thoroughly familiar with the lab work involved with the induction of the lac-operon (skilled at doing the work, not just the theory behind it). One of the big pitfalls noticed by this author is the lack of aseptic technique skills among most teachers. E.g.: it has been far too many times that this author cringed and shuddered as teachers assumed that "clean" means "sterile."

Anyway, here are a few links to help you renew your understandings of the theory behind the lac-operon.

  1. An overall view of gene control into which fits the lac-operon.
  2. This is about the kinetics of lac-operon induction, including some lab recipes.
  3. A tricky quiz to test your understanding of the lac-operon and the lab and its results.

With regard to a refresher course on lactase inhibition, there is none because this is a new finding as of 2001, and its teaching-lab instructions are adaptations of an original research project by Mireille Captieux (London, Eng.: A-levels project).

QUALITATIVE LEVEL (Class time = 45 minutes)

INTRODUCTION

In essence the two strains of bacteria are grown on the two different types of MacConkey agar plates. On the plain ones, the lac+ strain will grow as red colonies, while the lac- strain will grow but with non-red colonies. On the plates that have glucose, both strains should grow with red colonies. Using toothpicks, the students will lift all four types of colonies and test them for the presence of internal β-galactosidase to see if the appropriate cistron (lacZ) of the lac-operon is turned "on" or "off." The enzyme's presence is revealed when it degrades a structural analog of lactose called "ONPG" to make a yellow color. (β-galactosidase is also known as lactase.)

MATERIALS

PROTOCOL

  1. Do the first three steps 2 days before you meet with the students. Using a marker on the bottom of the various types of plates, draw a line that divides the plate in half. Put a "+" on one side of the line, and a "-" on the other side on all plates.
     
  2. Using a sterile swab transfer the "wild-type" to the "+" sides of all four types of plates: you need only draw the swab across the plate once or twice. Lines of bacterial growth will appear. Yes, you may use the same swab for all the plates, but do the swabbing of the lactose-only plates first so that you do not transfer any glucose to them.
     
  3. Usng a new sterile swab transfer the lacZ- strain to the "-" sides of all the plates, again doing the lactose-only plates first. All the plates may be incubated at room temperature, although storing them at a warmer temperature (up to 40C or 100F) will speed their growth. They should grow up to look like what is shown on the right.
     
  4. Have the students observe the growth and colors on the two types of MacConkey Plates. Dr. MacConkey cleverly devised these plates around 1906 based on the fact that E.coli will ferment a sugar to make a variety of acids which will turn the colonies red due to the pH indicator in the medium (read the bottle's label!). The fermentation is encouraged by the fact that there are toxins to aerobiosis in the medium. If the strain cannot use the sugar due to mutation, then it will grow using only the proteinaceous constituents and the release of excess ammonia will ensure that the indicator shows no acidity. In summary, the lacZ+ strain will be red on both the lactose-only and the glucose plates, while the lacZ- strain will be pale on the lactose-only plate (as it cannot use the lactose), but will be red on the glucose plate.
     
  5. And is there any β-galactosidase inside of the cells on these various plates? This will be determined by using the structural analog of lactose called "ONGP," which when cleaved by β-galactosidase forms a yellow color. Thus if one sees yellow, the enzyme must be present. No yellow means no enzyme. Because we are looking for yellow we cannot use the red or pink bacteria from the red MacConkey plates. We use the tryptone plates instead.
     
    1. Label four small glass testtubes +L, -L, +G and -G ("+" in lactose, "-" in lactose, etc., respectively).
       
    2. Add to each tube 1 ml tap water, 2 drops of chloroform, and two drops of the 0.1% SDS.
       
    3. Using toothpicks, the students scoop up small globs of the bacteria from the surface of the agar, and drop the toothpicks into their respective tubes.
       
    4. The tubes are then swirled violently for about 15 seconds (a Vortex mixer, figure to right, works nicely! Otherwise, with skill one can gallop their fingers to brush past the tube - try with water in tube first!). The SDS and chloroform emulsion break open the cell membranes, which then allows the ONPG to get inside to where the enzyme might be.
       
    5. Next add 4 drops of the ONPG solution to each of the tubes (here's a chance for you to talk about structural analogs).
       
    6. Watch for which tubes turn yellow (answer: see figure at right). Explain why "+G" did not turn as bright a yellow as did "+L". Explain why neither "-L" nor "-G" turned yellow. How come the tube "+G" was not yellow, while it grew a red colony on the MacConkey plate? You are now done with the lac-operon part of the exercise.
       
  6. Enzyme inhibition experiment.
     
    1. Each student has five test tubes labelled: X, Lac, Gal, Dex, Suc. (for "?", and lactose, galactose, dextrose and sucrose, respectively)
       
    2. Put 4 drops of ONPG into each tube.
       
    3. Add 1 cm of the appropriate sugar solutions from the side-shelf into the respective tubes ("X" is an "unknown" which you only know is nothing but water).
       
    4. After swirling the tubes briefly to mix, the students will add a drop of lactase solution to each tube and watch for color development. They should note the order in which the tubes turn yellow. If a sugar is an inhibitor, it should slow the reaction. Give whichever student speaks up about needing a control a whole pile of extra brownie points, and then mention what "X" is. The results should be almost immediately evident: X, Dex and Suc turn yellow almost immediately, indicating that Lac and Gal are inhibitors. Lactose would be expected to be an inhibitor because it directly competes with its structural analog - the ONPG. But what about galactose?
       
    5. Now you can talk about product or feedback inhibition. (The next quantitative section will reveal whether galactose is a direct competitor for the same active site on the enzyme [competitive inhibitor], or if it works in other ways such as by allosteric inhibition.)
       

QUANTITATIVE LEVEL

INTRODUCTION In essence the first part of this lab will be to time how long it takes for a culture of E. coli lac+ to turn on its lacZ cistron after the addition of inducer. For inducer, we will not use lactose, but rather "IPTG", which is another analog which lactase cannot breakdown. Thus the concentration of inducer remains constant. In the enzyme repression part, the initial rates of lactase digestion of ONPG will be measured in solutions doped with various concentrations of galactose. Those initial rates will then be graphed according to the method of Lineweaver and Birk.

MATERIALS

PROTOCOLS

  1. Kinetics of Lac-Operon Induction Class time = 2 hrs.
     
    1. On the day before you are to present this to the students, you should make up two 2-liter containers each containing 1 liter of STERILE broth containing 0.7% tryptone and a speck of thiamine (vitamin B1). After the medium has been cooled to room temperature, one of them should be inoculated with E. coli lac+, and then allowed to grow overnight with aeration. The other container will be used the next day.
       
    2. One hour prior to the students' doing the lab, add about half of the culture to the unused medium, and begin that aerating. The remainder of the culture may be discarded.
       
    3. Continue by following the steps given in this link. An example of results is shown to the right. From this you can see it took more than 3 minutes for the first β-galactosidase to form. For the next three or four minutes the concentration grew and then plateaued.
       
    4. Be ready to explain why there was a lag and why the enzyme didn't start forming immediately after the inducer was added. (Answer: both transcription and translation take time.)
       
  2. Ascertaining the Type of Inhibition Galactose Exerts on Lactase Class time = 2 hrs.
    This protocol will be an abbreviated on in which only one concentration of galactose will be used. Of course, if you wish a student to do a science fair project on this, many different concentrations should be used.
     
    1. Set up eight colorimeter tubes as shown.
       
    2. Run those without galactose first
       
      1. "Zero" one of the tubes in the spectrophotometer
         
      2. Add one of the following amounts of ONPG to the tube and swirl and reinsert the tube in the spectrophotometer and begin taking readings every 15 seconds for about 3 minutes. (Your "zero-time" was the moment you added the ONPG, your zero-time reading is 0.000. The amounts you will use are: 1 drop, 2 drops, 5 drops and 10 drops. When finished doing one of the ONPG concentrations, proceed to doing another until you have done them all.
         
    3. Now proceed to the tubes containing the galactose, and do similarly until you have recorded the increasing yellow of those four tubes.
       
    4. Determining the initial velocity of the eight various reactions
       
      1. Graph each of the eight data sets on bi-linear graph paper. You should get curves that look like this:
         
      2. Put a straightedge along the points that are at the beginning of the reaction thusly, and draw a line:
         
      3. Determine the slope of that line. If you use the same scales for all of the graphs, it eventually doesn't matter what units you use as they will all cancel out. Thus a slope might be so many centimeters rise per 3 minutes of time. These will be your initial velocities of reaction, Vo.
         
    5. Basic LB plotMaking your Lineweaver-Birk Plot, which in essence will look like this:
       
      1. On bi-linear graph paper, your horizontal axis (independent variable) will be the reciprocals of 1, 2, 5, and 10 drops. Thus: 0.1, 0.2, 0.5, and 1. Please don't forget the zero on the horizontal axis. (And how many drops does that represent? Yes, an infinite number, and that would the hypothetical maximum ONPG concentration.)
         
      2. Your vertical axis will be in centimeters.
         
      3. Convert all your Vo values to their reciprocals, 1/Vo
         
      4. Now plot the points of the four 1/Vo values of your tubes that contained NO galactose. Very lightly draw the best fit straight line through them in pencil.Label this line the uninhibited reaction, or normal.
         
      5. Now plot the points of the four 1/Vo values of your tubes that did contained galactose. Very lightly draw the best fit straight line through them in pencil.Label this line the inhibited one. (Did you expect this line to be above or below the "normal?") (Remember on this double reciprocal graph, higher means slower, and more rightward means more dilute. At 0,0 (the origin), the rate is infinitely fast, Vmax, at an infinite concentration.) (Who says you cannot plot infinity!)
         
    6. Interpretation of the Lineweaver-Birk Plot. If both the inhibitor and substrate (ONPG here) compete for the same place on the enzyme's surface, the constant concentration of inhibitor should become less and less effective as the concentration of the ONPG increases. If ONPG is in infinite concentration (at the vertical axis), then the inhibitor's effect should be insignificant. If this is the case, then the inhibited line should cross the normal line at the vertical axis ("C" in the following figure). However, if the inhibitor is doing something funny to another part of the enzyme and dialing down its speed a constant amount - say, to half speed, then no matter what the ONPG concentration, the speed will be half the normal, and the inhibited line will cross the vertical axis higher than will the normal (e.g.: graph "A").

      Allosteric vs competitive inhibition
       

    7. At this time you might want to look at your graphs, think about the errors built into this experiment and consider if you can really tell whether your lightly drawn lines are likely to be in the right places. Can you really discern if you experimentally derived graph looks more like "A" or "C". It might also be helpful for you to have an analogy of these types of inhibition in common street language. So look at this So look at this supermarket analogy.
       
    8. Clean up, or you will be thrown to the lions in the basement!
       

There are many different ways you can do the necessary experiments. To mention it again: it would be highly desirable for you to devise a plan and then write to this website outlining your plans. We will go over your plans, make suggestions and perhaps be able to offer you some helpful hints. Please remember to tell us your class's year in school.

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Structural ANALOGS: Tell the students that you will come back to this highly important topic as it pertains to many medicines and mutatgens.

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