TABLE OF CONTENTS OF THIS PAGE
(Clicking the first word in the item will take you directly to that section.)

1. Choosing the organism and genetic strains for the in vivo studies
2. Working on the genetic level
   1. Demonstrating how the various genetic strains express themselves under different conditions.
   2. Kinetics of induction and repression
3. Working on the enzymatic and metabolic levels
   1. Effects of inhibitors on the catabolism of lactose
   2. Effects of inhibitors on the concentration of lactase (β-galactosidase)

THE ORGANISM OF STUDY: What we shall be doing is feeding lactose to E.coli (a "genetic system"), while simultaneously subjecting the bacteria to various amounts of galactose. However we do not what the bacteria to be able to digest the galactose. To prevent that possibility, we needed to use a strain that is genetically incapable of using galactose. And we would compare this gal^- strain to a gal⁺ strain in the ability of the galactose to feedback inhibit the cells' lactase. So the following two strains are suggested:

Syndey Brenner's Accession Number	Yale's Accession Number	Genotype	Relevant Phenotype
MO	4995	F- λ- e14- relA1 rspL150(strR), spoT1 (see note)	lac+ gal+
M26	4996	same as above except it is also galE15	lac+ gal-
Note: "F- " indicates this is a 'female' cell (i.e.: without an F fertility factor) "λ-" indicates that this cell is not lysogenized with phage λ "e14- relA1 rspL150(strR), spoT1" all indicate that these cells have various abnormal ribosome functions

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How the Genotypes are Expressed

A "genotype" is what information is encoded in the genes, while a "phenotype" is how the organism appears. For example, you cannot tell much about the status of the lac-operon, for example, if the E.coli is growing in a medium containing no lactose and is consuming glucose. If you look inside the cell, you will not find any lactase. Initially you don't know if that is because the lac-operon is turned off, or if it is because the cistron of DNA that encodes lactase is damaged. (There are other possibilities for not containing lactase!) Switching things around, if the medium contains lactose, and you find that the cells contain lactase, you don't know if it due a normal gene's being turned on in a normal way, or if the on/off switch for that gene is "broken" in the "on" position and can never be turned off. We are now going to inspect these and other possibilities by growing our two selected E.coli strains on different media. Our fundamental medium will be called MacConkey agar "base", which contains agar, some Na₂HPO₄, pH indicators, bile salts (not relevant to E.coli), and some digests of protein. But there are NO digestible sugars present! Dr. MacConkey's rationale in developing this type of agar was that E.coli would either ferment a sugar or it wouldn't.

1. If it does ferment (note: 'ferment' means catalyze anaerobically) the sugar it would do it the only way E.coli 'knew' how to do it - by what is called "mixed-acid fermentation", which means that the sugar is converted into a number of different acids, such as acetic, formic, lactic, succinic, etc. First of all, the E.coli were obligated to ferment even if they were on an agar surface because one of the dyes poisons aerobiosis. The resulting acids would be in enough concentration and strong enough (formic is rather strong), to neutralize the rather weakly basic Na₂HPO₄ and drop the pH of the agar resulting in the turning of the methyl red dye to red. This dye is accumulated with the bacteria and the colonies become very red.
2. If it does NOT ferment the sugar, then the bacteria exclusively used the amino acids of the protein digest for their carbon sources. Not only were no "mixed acids" formed, but catabolism of hefty amounts of amino acids for their carbon content and making CO₂, results in a lot of ammonia being left over. This NH₃ dissolved in the medium turns it basic - completely the opposite pH direction! The colonies grow up as a light shade of pink.

(EMB agar is another type of sugar utilization agar, and works on much the same principle.)

Another neat thing about MacConkey agar is that it does contain that stuff called 'bile salts' - which are detergents used in our animal digestive juices. Because Gram-positive bacteria are very sensitive to detergents, and because the Gram(+) bacteria contain most of the desiccant and heat resistant sporeformers, this medium really doesn't need to be autoclaved - boiling to dissolve the agar is sufficient.

So all we need to do make several small amounts of MacConkey agar base, add various sugars into each of them, and then see what colors the strains grow on them. We are thus looking at their MacConkey phenotypes, right?

Supplies

• MacConkey agar "base" (i.e.: without any sugars - not even lactose)
  Most MacConkey agars sold have lactose in them - don't buy those!
• Powdered forms of several sugars - lactose, galactose, glucose
• Sterile swabs

1. Add 0.1 gm of each of the sugars into their appropriately labelled, dry petri plates. One plate, by the way, gets no sugar of any type (this is your negative control). Your positive control is the one with glucose since these strains both can utilize that sugar. (Really insightful students will make two more plates: one containing glucose and lactose, and the other containing galactose and lactose.)
2. Make four plates-worth of MacConkey agar 'base' according to the directions on the bottle. (Of course the insightful students make up enough for six plates!) Use tap water for best results (microbes need minerals to grow just like you do!). Bring that mixture to a boil. (All but the agar component will dissolve immediately upon addition of water. Agar dissolves at nearly 100c. Be careful when bringing it to a boil as agar solutions froth very easily. Swirl continuously while heating.
3. Pour in the hot agar solution so as to make each petri plate 2/3 full. Using different dry stirrers, get the sugars to dissolve and be mixed in well.
4. Waft a flame across the surface of the agars to burst any bubbles or foam that has formed upon pouring or stirring. Cover the plates and allow them to cool and solidify.
5. Immediately before applying the bacteria to the plates, go through the plate drying procedure.
6. Using the swab like a pen, dip it into some of the gal⁺ strain, and write a large plus on one side of the agar surfaces of each of the four plates - do the "NS" (no sugar) plate first.
7. Similarly write a minus on the other sides of the agar surfaces of the plates.
8. Incubate the plates for 24 to 48 hrs, and record which colors the bacterial strains grew on which plates. (Controls: both pink on "NS", and both red on glucose.) ((Psst: the 3-letter abbreviation for glucose is glc, as glu means glutamic acid! Guess what lac and gal mean.))

You now know which strain was capable of fermenting sugars. (Of course, the insightful students might not know whether one or both sugars were being fermented!) It would help to see if lactase is found inside of the variously grown bacteria. This we can do by making the cells permeable and seeing what they can do with an analog of lactose called ONPG (ortho-nitro-phenyl-galactopyranoside). If there is any of the enzyme lactase, the ONPG will be split into galactose and ONP, which is bright yellow. Thus "yellow results" mean lactase is in the cells, and no yellow means no lactase. Simple!

Supplies

• The plates made in the previous section. Better yet, a parallel set of growths on plates made with nutrient agar plus whatever sugars were used. The NA plates allow the growth of the bacteria without an accumulation of dyes that might confuse subsequent viewing of yellow colors.
• Flat toothpicks
• 0.1% SDS (sodium dodecylsulfate)
• Chloroform
• 12 ml ONPG solution (0.1 g ONPG/12 ml distilled water)
• 2 eyedroppers

Protocol

1. Using the toothpicks scrape up equal quantities of cells from each of the growths of the two strains on each of the types of plates.
2. Drop the toothpicks into appropriately labelled small testtubes containing 4.5 ml distilled water.
3. Vortex the tubes to detach and homogeneously disperse the bacterial scrapings into the water. Remove the toothpicks.
4. To each tube add 2 drops of chloroform and two drops of SDS.
5. Violently vortex each tube for 10 seconds. The chloroform and SDS form an emulsion that disrupts the cell membranes and permeablizes the cells so that subsequent addition of ONPG can enter the cells where lactase may exist.
6. Add 4 drops of the ONPG solution to each tube, swirl to mix. Note the development of yellow color in the tubes. Sharp eyes of the insightful students will also be able to discern which tubes turn yellow faster than others. The faster ones contained more lactase than the slower developing ones.
7. Your results should show that lactase is only produced if lactose is present, but the insightful students will see that if glucose is present with lactose then lactase is not present or only in very low amounts. The presence of galactose with lactose will work similarly for the gal⁺ strain, but not for the gal^-.
8. Create some hypotheses that explain your data.

Of course, you know that there are several cistrons in the lac-operon, and you might wish to study the gene's expression in various mutants. Here is a nearly complete list of mutants from which you may pick and choose: lacZ^-, lacY^-, lacA^-, lacR^- (lacI^-), lacR^c (lacI^c), lacO^-, and lacO^c. The students who are real gluttons for punishment might want to try strains with the lac-operon on the F-plasmid as E.coli lac^-/F-lac⁺ to see if being on a plasmid affects expression levels.

BRIEF NOTE ABOUT SOME OF THE MUTANTS: lacR^- (lacI^-) differs from lacR^c (lacI^c) because the repressor protein has two binding sites. One site binds to the DNA of the operator, and the other is an allosteric site that binds lactose (or IPTG) and inactivates the DNA-binding site. Thus you can have two different mutational expressions of the one gene depending on which binding site was affected. The same goes for lacO, which may either bind the repressor protein overly tenaciously resulting in the uninducible state, or it may not be recognized by the repressor protein and be "on" all the time (a state called "constitutive" - hence the small superscripted "c" in the genotype abbreviations). Most commonly lacO^c is found.

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Kinetics of the Induction and Repression of the Lac-Operon

The previous section considered cells that were grown for many hours in solutions of lactose. Physicists might call this the "steady state" expression of the lac-operon. In effect, the previous work tells us whether the gene was turned "on" or "off" under the prescribed conditions. What we want to do now is to determine how fast that "switch" works - either in the "on"-direction, or "off". Hence, the physicists would say that we are making a dynamic system, one that is not in steady state. Contrarian chemists like to call this "kinetics".

Kinetics of Lac-Operon Induction

In this, we shall be starting with cells that contain no lactase because they were growing in a medium devoid of any sugars - especially lactose. We will then add an inducer that will turn on the lac-operon, and then see how fast lactase accumulates in the cells. Of course, you can expect that several processes will be taking place - transcription of the lac-operon, and translation of the lacZ cistron yielding lactase.

At this point, the well-read student realizes that lactose sugar is not the best inducer of the gene because when the lactose is hydrolyzed it makes glucose and galactose and those, from the data of the insightful students in the previous section, cause a decrease in lactase. Just as ONPG is an analog of lactose, there is another, but non-hydrolyzable, analog called IPTG (isopropyl-thio-galactopyranoside). IPTG can thus be an inducer, but cannot be a substrate and be destroyed. Sounds like a winner!

The INDUCTION Experiment

..... The day before the students run their work, the instructor should make a 100 ml batch of sterile 0.7% tryptone broth in tap water in a liter flask; and also make a sterile 1-liter flask containing 400 ml of 0.7% tryptone. The 100 ml batch should be inoculated with a normal strain of E. coli lac+ and grown at 37c overnight with swirling. (Place the other flask at 37c also.) About 90 minutes before the students will need their cultures, dump the 100 ml overnight stationary culture into the other flask of 400 ml to re-envigorate the cells into growth again. (120 minutes was found to be too long before, and 60 minutes too short!)

1. Get together with your assigned group and have these ready:
   • You have lined up 17 little test tubes which have been labelled from "-1" to "15."
     The tubes should be set along one side of a WHITE rack, or a rack which has a piece of white paper on the bottom so that when you look down from the top into the tubes, you will see any colors that develop against the white background.
     
     
   • Add 2 drops of SDS to each tube.
     
   • Add 2 drops of chloroform to each tube.
     
   • Get your 'additive:' IPTG as a dry powder in a small microfuge tube.
     
   • Get your 24 ml of growing culture into your distinctively marked flask and place the flask into a clip in the shaker/waterbath, which has been set at 37c.
2. At time = -1 minute, take 1 ml from the flask and put it into tube "-1"; vortex the tube for 10 seconds; put back into rack. ..KEEP the flask forever ..SWIRLING! KEEP the flask forever WARM!
3. At time = "0", simultaneously (a) take 1 ml from the flask and put it into tube "0"; vortex 10 seconds; put back into rack; and (b) add your IPTG additive. ..KEEP IT SWIRLING! KEEP IT WARM!
   
4. Every minute that goes by take 1 ml from the flask and put it into the next tube; immediately vortex 10 seconds; put back into rack. ..SWIRL! ..WARM!
5. When you have finally taken the 15-th minute sample, send someone off to clean out the flask. ..Groups that leave dirty stuff behind will be dungeoned to be later thrown to very hungry lions!)
6. Add 4 drops of ONPG from a dropper bottle into each tube. ..Do this quickly but not sloppily. After each tube has gotten its four drops, have a partner swirl the tube to mix. (Often it is good to have several people adding drops to their assigned sections of the 17 tubes. This helps to get them all going at approximately the same time.
7. Put away all your bottles; clean out plastic droppers: ..REMEMBER THE LIONS!
8. Meanwhile your tubes might be slowly getting yellow. Keep an eye especially on those from 10 to 15 minutes. ..Once your higher numbered tubes get fairly yellow, ascertain the lowest numbered tube that shows even a trace of yellow. ..What does this mean? Why didn't tubes 0, 1, and 2 have any yellow?
9. If you have lined up all the tubes along the sides of a rack, the intensities will directly form visual sort of graph for your eyes.
10. You may wish to make this a rather precise quantitative experiment. If so, you will want to use a spectrophotometer (420nm) to determine the intensities of the yellows. You should then graph your data.
11. Meanwhile, clean out your tubes and invert them in your rack. (By this time the lions have gotten very hungry!)
    
12. Don't escape the lab yet. ..The instructor will go over the data. .. Be prepared to tell the significance of your group's findings, such as explanations for any lags.

Here's perhaps a hard question: Why didn't you merely add the IPTG, take just the one sample, vortex, and set the tube in the spectrophotometer and at timed intervals record how much yellow developed? (The answer: "This method will not work because....")