Bacteriology
for the Molecular Biologist
Bacteria are important tools to the Molecular Biologist. We use bacteria as little "factories" to make plasmid DNA or to express a particular protein we are studying. However, it is important to remember that bacteria are living organisms that do not always comply with our wishes. For example, if the protein you are trying to produce is harmful to the bacteria, you may find that it will only produce altered versions, or no protein at all.
The Escherichia coli (E. coli) bacterium is the most common in the Molecular Biology lab. There are many different strains of E. coli.Whenever a researcher introduces a change in the E. coli chromosome, she has created a new bacterial strain. It is important to know which strain you are working with and to understand how the mutations in the strain may affect your work.
Many Molecular Biology lab manuals and even some of the Biotechnology Company catalogues have lists of the common E. coli strains used in molecular biology. The strain and its genotype are given. The genotype will list all mutations in that strain. If a gene is not listed, you can assume it is the wild type. (what strain are we using in this exercise?)
Bacteria can grow in liquid media or on solid media. There are many specialized types of growth media. One that is often used for E. coli is called Luria broth, or LB. This is a rich medium that provides all the nutrients that the bacteria need to grow. If agar is added to liquid medium, it will solidify. LB agar petri plates provide a solid surface for bacterial growth. Thus, if you need a suspension of bacteria you can grow them in liquid media, but for individual colonies, you need agar plates. Certain molecular biology procedures will require either solid or liquid media.
When bacteria are inoculated into a fresh tube of liquid medium, they begin to grow by cell division. The bacterial growth curve has several distinct stages. See the lab book, pp. 24-25 for a description of the bacterial growth curve. The bacteria must be in exponential (or log) phase when we make them competent to take up plasmid DNA. This will be done later in the semester. When we want to isolate plasmid DNA, we often let the bacteria grow overnight ("overnight culture") and by the next morning they are in stationary phase. You should know the stages of bacterial growth and which stage is used for a particular molecular biology procedure for the midterm exam.
The optimal temperature for E. coli growth is 37*C (why?). E. coli in liquid medium can be grown in a 37*C incubator, but we usually use the shaking water bath adjusted to 37*C. The shaking motion keeps the bacteria in suspension and exposed to nutrients. In rich media (liquid) at the optimal temperature, E. coli have a doubling time of about 25 minutes. When we inoculate bacteria on petri plates, the petri plates are put in the 37*C incubator and the closed petri plates are always incubated upside down. The lid half rests on the incubator shelf and the agar half is on top. This is done to prevent condensation from dripping onto the colonies and spreading them around.
Bacteria are clonal. An isolated colony on a petri plate represents a population of bacteria derived from a single cell, and thus is genetically identical. This is very important to the Molecular Biologist. We must have confidence that all our DNA manipulations are performed on a genetically identical population.
Sterile technique is necessary when handling all bacteria. There are two important considerations to remember: (1) You must always protect yourself and your lab mates from being contaminated with bacteria. (2) You must also protect the bacterial strain that you are working with from contamination by other bacteria. All the supplies that you use for manipulating bacteria must be sterile, and after they are used they must be disposed of properly.
Common
Microbiology Manipulations:
Plasmids
Plasmids are small circles of DNA that replicate in the cytoplasm of bacterial cells, and thus are extrachromosomal elements. Many different plasmids have been isolated from bacteria. Some of them carry genes that are useful to the bacterial host, such as resistance to heavy metals. Other naturally occurring plasmids have no selective advantage that we can detect. Molecular biology depends on plasmids for many common techniques such as gene cloning and protein expression. The naturally occurring plasmids have been drastically altered to serve as cloning vectors. The plasmid is called the vector, while the piece of DNA to be cloned is called the insert.
Plasmid cloning vectors have one or more genes that code for antibiotic resistance. These antibiotic resistance genes produce a product (often an enzyme) that enable a bacterium that contains the plasmid to survive exposure to the antibiotic. The antibiotic resistance is a selectable marker for bacteria that contain the plasmid. If a bacterium contains the plasmid, it survives exposure to the antibiotic; if it lacks the plasmid, it dies. Antibiotics can be added to liquid or to solid media. They have different modes of action. The antibiotics that we use in lab are Ampicillin (Amp) and Kanamycin (Kan). The lab book has an explanation of their action and how resistance is achieved. See pp. 19-21.
Plasmids are present in the bacterial cell at a characteristic copy number. The number of plasmid copies per bacterial cell usually depends on which origin of replication is present on the plasmid. You should know the copy number of the plasmid you are working with. You may need to start with a greater number of cells to isolate enough of a low copy number plasmid to use in cloning. The majority of the vectors in use today are small plasmids, ranging from 3-5 kilobasepairs (kb), and they are high copy number.
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