STEP ONE: The Gene LacZ and Its Enzyme ß-gal'ase

To start, the students will comprehend that E.coli can have a gene that codes for an enzyme that can clip the sugar lactose in half. This is easily demonstrated by having the students look at a collection of petri plates containing MacConkey agar with and without selected sugars. Those without any sugar or are lacking the gene give negative test results. They can also look inside of those bacteria growing on lactose plates and find the enzyme ß-galactosidase ("ß-gal'ase") by using the ONPG test. There will be no surprises here for the students. All is as expected.

Refinements: you can talk about starting and stopping of transcription. There must be a "start" and a "stop" for your gene so the transcriptase will know what it is doing. So you add two short sequences to the beginning and end of the "lac" section. Rename the "start" to "promoter", that "stop" is a good word in most languages. (This gives you also an opportunity to talk about the world community of scientists and the need to be able to communicate - so words that are universal are best chosen to name things.)

Lab set-up: Three MacConkey plates are made: (Be sure that you buy MacConkey powder that contains NO sugars.) To one batch add no sugars; make another batch to about 0.5% lactose, and another batch to 0.3% glucose. Also make make three plates using nutrient agar: no sugar ("NS"), lactose, and glucose. (In both cases the NS and glucose plates are not shown to the students until they've gotten to Step Two, below.) Show the students the MacConkey agar reactions: E.coli are among the few bacteria that ferment sugars to acids. MacConkey agar contains crystal violet that poison electron transport so that the bacteria are forced to grow anaerobically (ferment), and the methyl red is a pH indicator that forms a red precipitate below about pH 4.5. So when E.coli ferments a sugar to an acid, a red colony grows up. Thus a lactose fermenting colony is red if lactose has been incorporated in the MacConkey agar.

Next introduce a bit of chemistry: the ONPG test. This is a neat way to trick an enzyme by using an analog. So discuss analogs, which are very important throughout biochemistry and in chemotherapy. This will be a real eye-opener for the students as it will show them a means by which they can tell if something is inside of a cell or not.

STEP TWO: Inducibility

Here is where inductibility is introduced. Some of the colonies on the above plates containing no sugars or glucose can be subjected to the ONPG test, and the students will discover that there is no ß-gal'ase in the cells. Yet those are the same strains that were grown on the lactose and did possess the enzyme! This is a very big item for discussion: the turning on and off of genes. You can talk about development and maturation of the students themselves. Then mention how hard it would be run tests to discover the mechanisms by which certain stages of development are initiated, and that by looking to extremely simple systems such as found in the bacterium, we can discover what is going on. So we need to refine the line diagram on the board to having some sort of switch. Let us call it an "operator" which must be somewhere very close to the promoter so that it will either allow transcriptase to start, or will prevent it from doing so. Of course, up to this moment, the students have no idea about how this operator might work. This allows you to introduce the idea that the "switch" is really more complicated than it looks. So you bring in the notion of a repressor protein that was encoded by the gene LacR. You can now bring in the sophisticated notion of "DNA-binding sites" - there are sections in proteins that bind to DNA, and this binding must be highly specific. Transcriptase binds to promoter regions in the DNA, while repressors bind to operator regions in DNA. Obviously there must be many similar promoter regions scattered around as each gene must have one at its beginning, but operator regions are generally unique.

Lab Set-Up: Suppose we have an inducible gene for an enzyme that degrades lactose. We put the wild-type and a Z^- on lactose+MacConkey, lactose+NB, and "ns"+NB The two grow on all the plates, and on the red plate, the Wild one make a red colony, and the Z^- does not. If we look inside of the cells using the ONPG test, then we see that the Wild has ß-gal'ase on the Lactose+NB, but not when grown on the "NS" plate. The Z^- strain does not test positive for ß-gal'ase in either case. The enzyme cannot be induced in any case! Must be that a functional cistron is missing. Everything seems simple. Thus the switch, lacR and lacO are also called into existence.

STEP THREE: Active Transport (LacY)

Just to check this out, let's take a look at a small collection of strains that do not make red colonies on the lactose red plate. So a lactose red plate is passed out with four non-red colonies (and a center red colony as control). The non-red colonies are lifted from a lactose plate and subjected to the ONPG test. Three show no yellow from the ONPG, but one does give yellow color. So this bug is inducible (no yellow from cells lifted from the NS plate). But for some reason didn't give red colony on red plate. Ask if any kids have any ideas what might be happening. Zero in on active transport: and that this mutant must not have the active transport mechanism. So there must be a second cistron in that operon - let's call it LacY, and its mutant would be termed LacY^-.

STEP FOUR: Regulators (LacO or LacR)

If the students take a look at several bugs that gave red colonies on the red plates, they find that there is one that gives a positive ONPG test no matter what sugars were or were not around. Discuss what this might be. A damaged O, or a damaged DNA-binding site on the R. Thus the students are seeing that even the regulator genes can be damaged (mutated). In this specific case, when the operator has a nonsensical sequence to which the lacR repressor protein cannot bind, we would call it a constitutive mutant (Lac O^constit.

Conclusion

These four steps allow the students to go from simplicity to complexity - each time being able to build confidently upon the previous well-understood 'simple' form.

STRAINS

About the bacterial strains that are being used. A great number of different ones are available. Below are given several that have historical significance. They are all derivatives of E.coli K-12 (remember the girl in the early 1920's who sat in row K seat 12 in the class that was taken over by a grad student named Joshua Lederberg. He was teaching because his boss, Fred Avery, was in the hospital for an extended time. The girl happened to be lucky and had isolated from the wild two different strains of E.coli that could exchange genes. One of those was the donor, and was eventually denominated "K-12". It was actually an F⁺ (possessing the F-plasmid). Many mutants have since been made from that strain, and some have even had their F-plasmids "cured" (gotten rid of). The strains that you may be using with your students are these. And I have appended their official numbers from the Cold Spring Harbor Lab (Watson is now director) on "LonGisland." Full genotypes are given:

CSH-25: Wild: Lac⁺ F- supF thiamine^- (requires trace of vitamin B1 to grow. Get some at health food store if I failed to give you any in the past. Less than a cubic millimeter is needed per liter. TRACE!

CSH-39: lacZ^-: F'-lacZ-pro⁺/lac,pro(deletion)thiamine^- Here you have a bug that has a defective lac-operon and a good proline operon inserted in the F-plasmid. The somatic chromosome is normal except that both the lac and pro operons are completely deleted. If ever you want to show sexduction (conjugation), cross this strain with one that is pro^- and you will quickly get a culture in which there are no pro^-. This strain can also be used to show segregation of alleles: grow it on medium with glucose and proline. The pro+ operon is not needed and if the plasmid gets lost, no matter to the cell. But by later testing it on medium without proline, those segregants will no longer grow. The mutation on the Z-cistron is due to an "amber" mutation - the wrong codon assignment for one of the amino acids. However, if this plasmid is inserted into cells that have a mutated tRNA for that same amino acid, the cistron can be read!

CSH-40: lacY^-: Almost exactly like CSH-39 except merely substitute lacY^- for 39's lacZ^-.

CSH-37: lacO^constit: Again like CSH-39 except merely substitute a damaged lacO. (This is a partially constitutive mutant - and might not give stunning yellows in the ONPG test when inducer is NOT present. But we saw, on the picnic bench that some yellow did occur.)

I think you might be getting the idea that while these strains are useful tools for teaching the rudiments of the lac-operon, they are also very powerful genetic tools for subsequent experiments - on the graduate school level.

PETRI PLATE PREPARATION

	INGREDIENTS
Sugars	Glucose and Lactose
Agars	Plain agar, and MacConkey Agar without lactose
Other	Tryptone powder, NaCl, thiamine (vitamin B1)

Plan so that each plastic petri plate needed should contain about 25 ml of agar, and each glass plate 30 ml. In the following are given recipes for 100 ml (4 plastic plates or 3 glass plates).

MacConkey Agar Plates: 5 gm of MacConkey Agar without lactose (or any other sugar for that matter!). If addition of a sugar is desired, add 0.3 to 0.5 gm of that sugar. Then add about 100 ml of TAP water (these are living things and need minerals just like you do!). Swirl to get everything in suspension, cover, sterilize. Swirl (shaking is a no-no! - might be super-heated; besides you will get a lot of foam) again as soon as taken out of the sterilizer to suspend the agar syrup at the bottom of the container.

Luria Agar: 0.7 gm of tryptone + a trace of vitamin B₁ (thiamine). (By a 'trace' is meant a speck about the size of a granule of table sugar.) If addition of a sugar is desired, add 0.3 to 0.5 gm of that sugar. And don't forget to add 2 gm of pure agar powder! Then add about 100 ml of TAP water. Swirl to get everything in suspension, cover, sterilize. Swirl (shaking is a no-no! as the liquid might be super-heated and become an uncontrollable gyser of boiling liquid; besides you will get a lot of foam) again as soon as taken out of the sterilizer to suspend the agar syrup at the bottom of the container.

ONPG Test: Dissolve about 200 mg of o-nitro-phenyl-ß-D-galactopyranoside (ONPG) in 25 ml of water. This will take nearly 30 minutes with a magnetic stirrer. It is now ready to use in droppers.

1. Add 1 ml of water to a test tube (small ones do nicely!)
2. Add 2 drops of chloroform to the tube and make sure that the water-immiscible chloroform sinks to the bottom of the tube. If it floats on the mensicus, it will evaporate and be gone within a minute).
3. Add 4 drops of 0.01% sodium dodecylsulfate (SDS) to the tube.
4. Using a toothpick or inoculating loop transfer a gob of bacteria from the surface of the non-red Luria plates and mix into the contents of the tube. Vortex the tube for 10 seconds ("One elephant, two elephants... ...ten elephants!") Use a tweezers to remove the toothpick.
5. Add 4 drops of the ONPG solution to the tube. DO NOT allow the dropper to touch the side of the tube lest you transfer some of the cells (possibly containing enzyme) back to the stock ONPG container.
6. Set the tube in a rack and watch it. If the enzyme ß-gal'ase is present a yellow color will slowly develop. (For a control and check on your reagents, use a speck of a lactase pill that you can buy in the supermarket. The yellow color will develop within seconds if all your reagents are properly made.)

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