Confirming the Phenotypes of E. coli Strains

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1 Confirming the Phenotypes of E. coli Strains INTRODUCTION Before undertaking any experiments, we need to confirm that the phenotypes of the E. coli strains we intend to use in the planned experiments correspond with their published genotypes. This is an essential preamble to any bacterial genetics project. More often than not, phenotypes in bacterial genetics are evaluated by plating cells on agar plates containing specially formulated growth media. Note that here we are using the techniques of classical genetics do not consult genotype directly, but only indirectly, via phenotype. Directly verifying the genotype involves Molecular Biology rather than Genetics. This means using techniques such as cloning, DNA sequencing, PCR, or Southern analysis. While these methods potentially provide a more definitive characterization of a strains genotype, they all generally involve more time and money than straightforward phenotypic characterization. So, it's a trade-off. STRAIN DESCRIPTION Here is a list of the 4 strains we will check, and a description of their genotypes and properties. Following that there will be a description of the growth media. We want to help guide you through the process of putting these two sets of descriptions together in your heads so that you understand what each strain should do on each of the media. UCSC Stain# SC071 SC142 This is MG1655, the standard "wild-type" for the E. coli K-12 lineage. It is F - (λ - ), but otherwise should have the "wild-type" genotype for all genes of interest to us in the course. Δ(gpt-lac) SC109 Δ(gpt-lac) rpsl (=stra1) SC138 Δ(gpt-lac) Tn5 F' proa, prob laci, lacz, lacy Page 1 of 6

2 Δ(gpt-lac) was described in the nomenclature exercise. The rpsl in strain 109 is the classic mutant allele originally designated stra1; this was also described in the nomenclature exercise. SC138 is a more complicated beast. For starters, it has a copy of the transposon Tn5 inserted in the chromosome at an unknown location. You will need to read the posted references on Tn5 to find out how it will affect the behavior of SC138. SC138 also has an F' plasmid. F' plasmids are derivatives of the original F conjugal plasmid that has acquired one ore more genes from the E. coli genome. In this F', the plasmid has carries a large region of the E. coli genome that extends from the genes proa and prob ( ) to the Lac Operon ( ). Description of Media Broth Media vs. Minimal Media Bacterial growth media fall into 2 broad categories, BROTH Media and MINIMAL Media. It is critical to understand the distinction between these types in order to interpret your results. BROTH media are generally prepared from aqueous extracts of cells or tissues, combined with hydrolyzed proteins. Thus, they provide a rich variety of organic molecules (amino acids, nucleotides, carbohydrates, etc.) that can be used as carbon and energy sources for bacterial growth. They support rapid growth, even of E. coli strains with mutations that eliminate some of the metabolic characteristics of the wild-type. They are also easy to prepare. The drawback is that their molecular composition is not only complex, but undefined. the broth medium we use is medium, the ubiquitous, standard E. coli growth medium in molecular biology. MINIMAL media may contain only a single type of organic molecule added to a solution of inorganic salts (ammonium, phosphate, sulfate, etc.). Growth of a strain in a MINIMAL medium implies that the strain is prototrophic, meaning that it has a complete set of biosynthetic pathways, and that it can metabolize the particular carbon source provided (frequently glucose or some other sugar). Auxotrophic mutants cannot grow in un-supplemented MINIMAL media because they lack at least one complete biosynthetic pathway. For example, an E. coli mutant strain lacking a gene for an enzyme in the histidine biosynthetic pathway has a His - phenotype and will not grow in Glucose Minimal Medium unless the medium is supplemented with histidine. Minimal media are more trouble to prepare, and do not support as rapid growth as broth media, but they have the great advantage that their molecular composition is defined. A defined medium is one whose composition is known precisely at the molecular level. Defined media are almost always used in metabolic, nutritional or physiological growth experiments. The media used, and the plate code used to identify them are: Broth Media: + Streptomycin I + Rifampicin II + Kanamycin K + Triclosan Ts + X-Gal X* Minimal Media: Minimal Glucose I I Minimal Glucose + Proline I I P Minimal Lactose + Proline I II P Page 2 of 6

3 *X-Gal, an artificial, chromogenic substrate for the enzyme beta-galactosidase (the lacz gene product). X-Gal is colorless, but its hydrolysis product is blue. Therefore, only Lac + strains produce blue colonies on X-Gal medium. This is the substrate commonly used in detecting recombinant plasmids in classic molecular cloning. You should print a copy of the Agar Plate Code from the web site and keep it in your notebook. Page 3 of 6

4 PROCEDURE DAY 1 We provide you with overnight cultures of the 4 strains. Overnight cultures will be a typical starting point for many of our exercises. During the growth of these cultures (15-24 hrs) one or more essential nutrients become/s depleted, and the growth rate falls to an insignificant level. That is, the cell density in an overnight culture probably represents the carrying capacity of the medium, or, as a microbiologist would say, the culture is in stationary phase. For E. coli cultures grown in Medium, the cell concentration in an overnight culture is usually about 2 X 10 9 cells/ml. Compare the appearance of your overnight cultures to the appearance of sterile Medium. After your overnight cultures sit undisturbed in the bench for an hour or so look at them again. How does the appearance of the culture change? What does this mean? Dilute 10 ul of each overnight culture into 1 ml of sterile saline (0.9% NaCl) and mix thoroughly. (In 4 separate tubes, that is. Don't mix all the different strains together in one tube.) This represents a 10-2 dilution. 0.9% NaCl (sterile saline) is a generic vehicle for the suspension and dilution of E. coli. No nutrients are provided for growth, but isotonic conditions prevent the osmotic effects that might injure the cells if they were diluted in di water. Get all your agar plates and label them on the bottom according to the diagram below. Transfer 5 ul drops of each diluted overnight culture onto the agar plates. (Do one strain on all plates using the same micropipette tip. Then change tips and do the next strain.) Streak the droplets lengthwise on the plates with a sterile glass rod. See the diagram below. (You can streak the same strain on all the media before you need to re-sterilize it.) Allow the plates to sit on the bench until all free liquid has been absorbed into the agar. Incubate the agar plates upside down in a 37 C incubator. Wild-type E. coli produce visible colonies (>0.5 mm dia.) on plates overnight. Formation of visible colonies on minimal plates requires incubation for hours. Page 4 of 6

5 PROCEDURE DAY 2 Before you come to class, complete the table below by filling in your predicted result for the 5 strains on each medium. Describe your prediction in terms of what you would see when you look at the plate. Be sure to define any abbreviations or symbols (i.e. + = confluent growth etc.). Make a copy of the completed table and give one to the TA before you examine your plates. Keep the other copy for reference. Don't fret about "getting it wrong", just take your best shot and, certainly, ask us questions that will help you figure out what should be happening. Strain Strep Kan Rif Tric X-Gal Minimal Glucose Minimal Glucose Proline Minimal Lactose Proline SC071 SC142 SC109 SC138 Now, examine your plates and carefully record your observations in your notebook. Words and pictures can both contribute to robust results. Page 5 of 6

6 ASSIGNMENT 1. [10 pts.] Completed table with your predicted results (submitted before making your observations.) 2. [20 pts.] Completed the table with the actual, observed result for the 4 strains on each medium. Note: The results may not be as simple as "Growth" vs No Growth". 2. [40 pts.] Write an organized discussion of whether or not the phenotypes of the strains, as demonstrated by growth on the agar plates, are consistent with their reputed genotypes. In particular, focus on an analysis of any discrepancies between your results and your original predictions. This is an opportunity to use the strategy of first creating an outline of what you wish to convey, so that you can avoid a "stream of consciousness" effect. 3. [10 pts.] Approximately how many E.coli cells were in each 10 ul drop spotted on the plates? 4. [10 pts.] Why did we dilute the overnight cultures in sterile saline before plating them? Wouldn't it have been faster to just plate them directly? Page 6 of 6

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