SECTION I1 BACTERIA AND CLONINGBACTERIAL PROPAGATION INTRODUCTION: There is heavy utilization of microbiologicaI methods in molecular biology, from molecular cloning of plasrnid and phage DNA in Esckrichia coli to transformation of plant cells with Agroblacterium tumefaciens. In the next several weeks we will be inserting foreign DNA (from plants, fungi, or whatever other source you want to use) into a plasmid (pBR325), that will then be introduced into and replicated by E. coli cells for later study. I I Th.e first step in this process is to select and grow the proper strain of bacteria. We will use E. coli, strain HB101, cells. These cells have been developed to be extremely disabled with respect to their ability to survive outside the lab. Next, you should choose the vector that is the best suited for the purpose at hand. For DNA pieces up to 15-20 kb in size, plasmids can be used (although pieces below 10 kb are much easier to clone than larger pieces). For fragments up to 25-30 kb, bacteriophage lambda can be used. For fragments up to 50 kb, cosmids can be used, althpugh pieces of this size are often difficult to clone. For DNA sequencing, single stranded DNA bacteriophage MI3 is generally used. In each of the above categories, there are many different commercially (and academically) available choices. The plasmids we will be using (based on pBR322, pBR325, and their derivatives) have genes that confer resistance to the drugs chloramphenicol, ampicillin and tetracycline to the host bacterium. [Chloramphenicol binds to the 50s ribosomal subunit and halts protein synthesis, ampicillin is a derivative of penicillin and inhibits cell wall formation, tetracycline binds to the 30s ribosomal subunit and stops protein synthesis.] There are some unique restriction enzyme sites in each of the resistance genes that make selection of recombinant molecules possible. For example, we will be cloning into the EcoRI site located in the gene that confers chloramphenicol resistance to the host. All of the bacteria that carry an uninterrupted gene are able to grow on media with chloramphenicol. how eve^, the bacteria whose chloramphenicol resistance gene has been interrupted by the insertion of a foreign DNA fragment, will not be resistant to the drug. They will, however, be resistant to ampicillin and tetracycline. We will also use plasmids that carry the gene for p-galactosidase. The host Bacteria have been engineered to lack this coding region, and only contain the a-galactosidase coding region. Since both parts are necessary to produce functional galactosidase enzyme, only bacteria that have been transformed with a functional p-galactosidase (on the plasrnid) will exhibit galactosidase activity. Since the P-galactosidase complements the a-galactosidase, the process is known as "a-complementation." The activity of galactosidase can be made visible by the addition of a dye (5-bromo-4-chloro-3-indoyl-p-~-galactoside, also known as X-gal) that is colorless (to slightly yellow) until acted on by galactosidase, after which it forms a blue precipitate. Thus, a bacterial colony exhibiting galactosidase activity appears blue, and one that lacks functional galactosidase appears white (or creamy). There are many different media for growing bacteria. We will use one of the most common media, known as LB (Luria-Bertani) medium. This first lab period will be spent preparing the liquid medium and agar plates for the growth of the bacteria. Each group should prepare three bottles each of liquid LB medium and LB mediumwith agar. After autoclaving, followed by about 5-15 minutes of cooling at room temperature, the drugs are added and the plates are poured. The addition of X-gal to the "X-gal/LBAM plates should be done an hour or so prior to spreading of the bacteria onto the plates. X-gal is not stable in solution at room temperature. The following are the amounts of liquid medium and the number of agar plates that you will need for the next few weeks: Liquid medium, 150 rnl LB 175 ml LBA (LB with ampicillin) 175 ml LBC (LB with chloramphenicol) For plates (all with agar), 150 rnl LB (for six plates) 350 ml LBA (for fourteen plates) 100 ml LBC (for four plates) For recombinant DNA work you should be aware that there are restrictions on lab setting, containment, disposal and type of organisms that can be used. Here are some general guidelines for our work in this lab: 1. The doors and windows in the lab must be closed when using any recombinant molecules or bacteria containing the recombinant molecules. 2. All unused recombinant molecules must be securely stored (a freezer or. refrigerator is fine). 3. Any recombinant molecules or bacteria must be autoclaved before disposal. 4. DNA from pathogens of plants or animals may not be used for these exercises unless specific permission is obtained. 5. Use of human DNA or human pathogen DNA is forbidden, unless you obtain special U.S. government authorization. STEPS IN THE PROCEDURE: PART I: MAKING MEDIA AND AGAR PLATES: I. Measure out and dissolve the ingredients for the LB and LB with agar, as listed above. These should be in bottles that have caps. [NOTE: The agar will not go into solution until autoclaving.] Be sure that the bottles are less than half full or the liquid, will boil out of them. Also, leave the caps loose on the bottles or they will explode in the autoclave. 2. Autoclave all solutions on a 20 minute liquid cycle.3. While the LB is autoclaving make the stock solutions of ampicillin and chloramphenicol. Make only enough to add to the liquid medium and to the agar plates. Keep these in the freezer until they are added to the medium. 4. Prepare the sterile hood (in room 432 Illick) for use. If they are not already on, turn on the fan and the fluorescent lamp (not the UV lamp). If the Bunsen burner is on, turn it OFF! Squirt some 80% ethanol onto the hood table and use a tissue to wipe the entire surface of the hood table with the ethanol. Leave the light and the fan on until you ate ready to use the hood. 5. When the LB is out of the autoclave swirl the containers to assure complete mixing, especially for those containing the agar. [If someone else is using the hood, or for some other reason you cannot pour the agar-containing medium, keep it warm in a 65 "C water bath until ready. However, if you plan the pour the plates on a different day, place the container into a refrigerator and