The bacteria share this vital information by passing it among themselves in the form of genes in plasmids. Hence, the natural function of a plasmid is to transfer genetic information vital to the survival of the bacteria. It is this characteristic of plasmids that is exploited for use in transformation.
For bacteria to take in a plasmid, they must first be made "competent" to take up DNA, which won't normally pass through a bacterial cell's membrane. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium.
DNA can then be forced into the cells by the procedures followed during this experiment. In this activity, students will use a strain of E. One gene codes for a green fluorescent protein GFP and the other codes for ampicillin resistance. The source of the GFP gene is the bioluminescent jellyfish Aequorea victoria. The ampicillin-resistance gene allows us to select which of the E. Figure 01 - Click to Enlarge. This combination of genes was chosen because the protein produced from this combination turns bacteria yellow-green, even in normal light.
If you expose the colonies to a UV light, they also fluoresce. The plasmid also contains the antibiotic resistance gene to allow growth in the presence of ampicillin. The plasmid DNA should be kept in the refrigerator until it is aliquotted for students. Day 1: Follow directions on the E. Aliquot microtubes with just over 1 ml of 50 mM CaCl 2 to each be shared by two lab groups. Obtain enough crushed ice to fill foam cups or beakers for each lab group.
Aliquot one microtube with 1. When all of your classes have finished using the streak petri plates, open the dishes and immerse in bleach solution for at least 20 minutes. Following sterilization, pour off liquid, put dishes in plastic bag, and throw away in regular trash. As previously noted, the E. However, it is very important to follow the guidelines below for handling of the bacteria and disposal of waste to ensure that the E.
In molecular biology, transformation refers to a form of genetic exchange in which the genetic material carried by an individual cell is altered by incorporation of foreign exogenous DNA. This foreign DNA may be derived from unrelated species and even other kingdoms, such as bacteria, fungi, plants or animals, which would otherwise be inaccessible to an organism.
Bacteria and yeast have been transformed with human genes to produce proteins that are useful in treating human diseases and disorders e. Some bacteria have been modified such that they are able to digest oil from accidental spills. Bacteria are single-celled organisms that can easily pass information between one another and thus changes in genetic make-up are rapidly passed on to subsequent generations.
Transformation is usually more difficult with multicellular organisms, such as plants, in which it is necessary to either alter many cells with the new piece of DNA or to alter just a single cell and then induce it to produce a whole new plant. Genetic transformation of plants and other organisms does occur naturally.
The bacterium you will be transforming, E. Its genetic material consists mostly of one large circle of DNA million base pairs mbp in length, with small loops of DNA called plasmids, usually ranging from 5,, base pairs in length, present in the cytoplasm. It is these plasmids that bacteria can transfer back and forth, allowing them to share genes among one another and thus to naturally adapt to new environments. The ability of bacteria to maintain these plasmids and replicate them during normal cell multiplication is the basis of cell transformation.
In this experiment, green fluorescent protein GFP from the bioluminescent jellyfish Aequorea victoria has been incorporated into a plasmid along with a gene for resistance to the antibiotic ampicillin. The GFP is actually located in discrete spots around the bell margin of the jellyfish and will fluoresce under certain conditions When inserted into a plasmid and used for the transformation procedure, the transformed bacteria will express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow green under ultraviolet light.
This plasmid contains an ampicillin-resistance gene in addition to the GFP gene. Ampicillin is an antibiotic and works by preventing E.
When the ampicillin-resistance gene is present, it directs the production of an enzyme that blocks the action of the ampicillin, and the bacteria are able to survive. Bacteria without the plasmid and, hence, the resistance gene are unable to grow on a plate containing ampicillin in the medium, and only the transformants will survive.
GFP is also used in research as a reporter molecule. It can be linked to the protein that you are interested in studying, and this protein can then be followed through changes in expression of the linked GFP. Review the safety instructions with your teacher to ensure that you know how to handle the cultures and equipment safely.
Clean the lab benches with the bleach solution and remember to wash your hands before leaving the lab. Stop Point: The tubes can be refrigerated overnight and removed just prior to the beginning of the next lab. If you will not finish the lab today, give the tubes to your teacher for overnight storage. Clean up: Place used loops etc in the bacterial waste container. Spray workspace with bleach solution and wipe with paper towels. Wash hands before leaving lab. Note: Factors influencing transformation efficiency include technique errors, the temperature and length of the incubation period, the growth stage of the cells, and using the correct mass of plasmid DNA.
The protocol above has been modified from UC Davis. The website below provides information and pictures of the transformation and regeneration of a tomato explant using Agrobacterium tumefaciens to carry the engineered DNA into the tomato cell. Log In Bookstore Join Renew. It looks like your browser does not have JavaScript enabled.
Please turn on JavaScript and try again. Activity 4: Transformation of E. Page Content. Gloves and safety glasses are to be worn at all times during this experiment. Keep nose and mouth away from tip end when pipetting suspension culture to avoid inhaling any aerosol that might be created. Introduction Transformation of cells is a widely used and versatile tool in genetic engineering and is of critical importance in the development of molecular biology.
Figure 01 - Click to Enlarge Objectives Understand recombinant DNA techniques, in particular the transformation procedure using the heat shock method. Understand the uses of marker or reporter genes in molecular biology experiments and how to screen for a gene of interest. Understand that DNA can be transferred to another organism and therefore change the observable characteristics of that organism.
Some find it too rich, and others find it deficient. The nutrient in this is beef broth, and some extracts from yeast. Sabouraud Agar Used for fungi and has a low pH that will kill most bacteria. It contains gentamicin, which is a aminoglycoside antibiotic. Gentamicin can also treat many different types of bacterial infections, particularly Gram-negative infection. Thayer-Martin Agar Chocolate agar designed to isolate Neisseria gonorrhoeae , also known as "gonococcus," which is a species of Gram-negative bacterium responsible for the disease gonorrhoea.
Tryptic Soy Agar A basic medium used for culturing many kinds of microorganisms. Tryptic soy agar is mainly used as an initial growth medium for the purposes of: observing colony morphology, developing a pure culture, achieving sufficient growth for further biochemical testing, and culture storage. XLD Agar Xylose lysine deoxycholate agar. It is used for the culture of stool samples, and contains two indicators.
It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters appear yellow. Preparing Bottled Agar and Plates 5 Pre-experiment: Keep sterile Petri dishes closed until ready to pour agar into them.
Air-borne contaminants can easily invade an open Petri dish. Although pre-poured agar plates are available, one can make agar plates from tablet, powdered, or bottled agar by following a few simple instructions. Agar kits usually come with detailed instructions on how to prepare plates, and below are sample procedures for reference.
When in doubt, be sure to clearly read the instructions and ask for help if needed either consult a teacher or call the technical help line of the agar kit supplier. The formulation for LB Luria Bertani agar is: 9.
If using tablets, dissolve 10 tablets per ml of water. For agar powders, dissolve by microwaving, 6. Stack agar plates upside down in the refrigerator. Do Not Freeze! The purpose of placing the plates upside down is to prevent condensation from dripping down onto the agar surface which could then facilitate movement of organisms between colonies.
Place each Petri dish inside a zip lock bag to prevent drying out and to control odors. Turn the plates upside down and put them in a warm place. Bacterial growth should start to become visible in days. For those growing bacteria at home for example, investigating bacteria growth at various places around the house , you may use a homemade "light bulb incubator" in place of a laboratory incubator.
Once the Petri dishes have been taped shut, they should not be opened again. All microorganisms grown during the experiment should be killed before discarding. The best way to dispose of bacterial cultures is to pressure sterilize them in a heat stable biohazard bag. If autoclaves or pressure cookers are not available or large enough to make this convenient, an alternative is to bleach the plates.
Let them sit and soak overnight in the bleach solution before disposing of them. Please note that the bleach solution is corrosive and needs to be thoroughly removed afterwards. In addition, the plates can be incinerated if access to an incinerator is available. Dorland, W. Dorland's Medical Dictionary. Please follow the item description at the bottom, next to the catalog number, and not the picture caption, which says non-nutrient agar.
Menu Science Projects. Project Guides. View Site Map. Science Projects. Grade Levels. Physical Science. Earth and Environmental Science. Behavioral and Social Science. Explore Our Science Videos. Harvest Water from Fog Science Project. Comprised of sheep blood that provides the X and V factors necessary for Haemophilus growth, this is a nutrient medium which is used in culturing fastidious organisms such as Haemophilus species and Neisseria.
LB Luria Bertani Agar. A subtype of nutrient agar, this is the general medium for microbiology studies and may be used for routine cultivation of not particularly fastidious microorganisms. This is an agar upon which only Gram-negative bacteria can grow.
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