_________________________
Exercise Name 10: Behavior of lac Mutants on Indicator Plates
[Adapted from MILLER, JEFFREY H., Experiments in Molecular Genetics, Cold Spring Harbor NY: Cold Spring Harbor Laboratory, 1972, pp. 4755.]
INTRODUCTION The lac operon of Escherichia coli (Fig. 1) has been a favorite example of genetic regulation since it was first described (see JACOB & MONOD, 19611).
lacI 5'
Figure 10-1. The lac operon. The lacI cistron encodes a repressor protein which is transcribed constitutively and binds to the operator (O) region of the operon, blocking transcription initiated at both P1, a poor promoter, and P2, a mediocre promoter. Some leakage is allowed, and small amounts of the products of the three structural genes are always found in the cell. Cistron lacZ encodes -galactosidase, lacY encodes a permease, and lacA encodes thiogalactoside transacetylase. When lactose is in the cell's environment, some penetrates the cell by means of the permease and is converted to allolactose by -galactosidase. Allolactose binds to the lacI gene product and prevents its binding to the operator region, thus enhancing transcription of the structural genes. P1 is converted to a good promoter in the presence of a complex of 3',5'cAMP and cAMP receptor protein (a starvation signal) and transcription is greatly enhanced under these conditions.
P2
P1
O
lacZ
lacY
lacA 3'
The phenotypic behavior of various ing colonies on various indicator plates taking place (and in some cases, roughly quantitatively for -galactosidase activity. we will do the latter.
lac mutants can be observed either by growthat signal whether lactose fermentation is how much), or by directly assaying the cells Today we will do the former and next week
FRANCOIS, & JACQUES MONOD. Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology 3: 318356, 1961.
1JACOB,
Monday, February 12, 2007
Page 63
1:11:33 PM
BIOLOGY 310: PROKARYOTIC AND VIRAL GENETICS LABORATORY MANUAL EXERCISE 10: BEHAVIOR OF lac MUTANTS ON INDICATOR PLATES In addition to enabling us to score different phenotypes simply by observing colony color, the proper use of indicator plates makes possible the screening of large populations for a wide variety of mutants. Some indicator plates (EMB, tetrazolium, and MacConkey) make use of the pH differences between colonies which metabolize specific carbohydrates and colonies which do not. Others, such as Xgal, employ specifically prepared substrates which release a colored dye when hydrolyzed by the specific enzyme being studied. These substrate dyes are either dissolved in the medium itself, or else sprayed onto the surface of the plate after the colonies have grown. The two types of indicator plates you will be using this week are:
MacConkey (lactose) plates: Lac+ colonies are dark pink and Lac colonies are white; intermediate levels of fermentation, such as that of cells fully derepressed with isopropylthiogalactoside (IPTG) but incapable of making functional permease are indicated by a lighter shade of pink. Xgal (glucose) minimal plates: 5-Bromo-4-chloro-3-indolyl--D-galactoside (Xgal) allows the detection of various amounts of constitutive galactosidase inside E. coli colonies. The Xgal, which is not an inducer of the lac operon, is colorless itself. However, when hydrolyzed by galactosidase, this compound releases deep blue 5-bromo-4-chloroindigo. Thus i colonies appear deep blue while i+ colonies appear white.
You will be given four strains of E. coli of known genotype and two strains of unknown genotype. You will observe the behavior the of six strains on MacConkey (lactose), MacConkey (lactose) + IPTG, and Xgal (glucose) minimal plates; you will then identify the genotypes of the unknown strains with regard to their abilities to ferment lactose.
STRAINS ATCC 23721 Escherichia coli EMG 7 or 2000. Genotype: K-12 thi. Deposited by R. C. Clowes. ATCC 23722 Escherichia coli EMG 8 or 20S0. Genotype: K-12 lac (i+zy+) thi. Deposited by R. C. Clowes. ATCC 23723 Escherichia coli EMG 9 or 3300. Genotype: K-12 lac (iz+y+) thi. Deposited by R. C. Clowes. ATCC 23724 Escherichia coli EMG 10 or C600. Genotype: thi-1 thr-1 leuB6 lacY1 tonA21 supE44 (lambda). Deposited by R. C. Clowes.
Monday, February 12, 2007
Page 64
1:11:33 PM
BIOLOGY 310: PROKARYOTIC AND VIRAL GENETICS LABORATORY MANUAL EXERCISE 10: BEHAVIOR OF lac MUTANTS ON INDICATOR PLATES TABLE 10-1. EXPECTED BEHAVIOR OF KNOWN STRAINS AFTER 24 HOURS OF INCUBATION Lac i+z+y ATCC 23724 Ever so faintly pinkish with yellow halo Faint pink with yellow halo at 24 hours, deep pink at 36 hours Ever so faintly blue i+zy+ ATCC 23722 White with yellow halo White with yellow halo Lac+ i+z+y+ ATCC 23721 Deep pink iz+y+ ATCC 23723 Deep pink
MacConkey (lactose) MacConkey (lactose) + IPTG
Deep pink
Deep pink
Xgal (glucose) Minimal
White
Faintly blue
Blue
MATERIALS, DAY 1 One overnight each of ATCC 23721, 23722, 23723, 23724, and the two unknowns Six plates of LB agar
PROCEDURE, DAY 1 1. Streak ATCC 23721, 23722, 23723, 23724, and the two unknowns on six different LB plates such that single colonies will be obtained. Incubate the plates overnight at 37 C.
MATERIALS, DAY 2 50 sterile toothpicks, wrapped in aluminum foil One plate of LB agar
Monday, February 12, 2007
Page 65
1:11:33 PM
BIOLOGY 310: PROKARYOTIC AND VIRAL GENETICS LABORATORY MANUAL EXERCISE 10: BEHAVIOR OF lac MUTANTS ON INDICATOR PLATES PROCEDURE, DAY 2 1. Using the sterile toothpicks pick individual colonies of ATCC 23721, 23722, 23723, 23724, and the two unknowns onto alternating rows on an LB plate. Put a reference mark somewhere on the rim of this master plate, and incubate it at 37 C overnight.
MATERIALS, DAY 3 One LB agar plate One plate of MacCo...