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.
Become familiar with sterile technique and decontamination procedures that are used to handle bacteria. Learn how to calculate transformation efficiency. Store these plates in refrigerator until ready for use. If you choose to make your own plates, the directions for ml of LB agar for plates are as follows: Measure ml of LB broth into glass beaker or flask, add 7. Boil the solution on a hot plate or in the microwave two second intervals with agitation between. But the real reason was to compare protein expression systems in E.
The plasmid also contains resistance to ampicillin, an antibiotic that inhibits the cell wall formation in gram-negative bacteria like E. Here is a map of the arabinose induced pGLO plasmid :. Arabinose is a simple plant sugar and is used as food by bacteria like E. The bacterial genes for arabinose digestion are not expressed unless arabinose is present. No need to waste all that energy on nothing, right? When arabinose is present, however, these genes get turned on and arabinose is broken down.
When arabinose runs out, the genes turn off. Three structural genes araB , araA , araD or BAD encode for enzymes used in the digestion of arabinose in bacterial cells. Another gene called araC produces a protein that regulates the expression of BAD. The a raC protein also acts to negatively control its own synthesis. In other words, the araC protein binds to its own operator and prevents more araC transcription.
The presence of both the araC protein and arabinose cause the expression of BAD. Arabinose binds to the araC protein and causes a change in its shape, which then allows both to bind to the regulatory region known as the activator site. These three genes can now be transcribed and the resulting RNA can be translated into protein. Thus, araC negatively controls its own expression and positively controls BAD.
Here is a summary figure of the arabinose operon:. Comparison of original arabinose operon with the GFP modified operon. In its native environment, GFP fluoresces in the deep sea jellyfish, Aequorea victoria. Incredibly, GFP retains its fluorescent properties when cloned and expressed in E. These extensions link two of the most commonly used techniques in biotechnology labs: transformation and protein purification.
Purification of a protein depends on a its chemical or physical properties, such as molecular weight, electrical charge, or solubility. GFP can be separated from others by its size using electrophoresis, and it is extremely hydrophobic, which enables its purification using hydrophobic interaction chromatography HIC.
When placed in a buffer containing a high concentration of salt, the HIC matrix selectively binds hydrophobic GFP molecules while allowing the bacterial proteins to pass right through the column. Then, simply lowering the salt concentration of the buffer causes GFP to elute from the column in a purer form. Students can explore these separation techniques by growing transformed bacteria in liquid culture to grow overnight, then lysing the cells to release their contents.
The unique fluorescent property of GFP allows real-time monitoring of extraction and purification, modeling key processes used in biotechnology to produce and purify designer proteins with commercial or research value. Use these short, instructional videos to enrich lessons about bacteria, bacterial transformation, and the green fluorescent protein GFP.
Investigate the functional elements of pGLO bacterial transformation, including heat shock, antibiotic selection, promoters, and satellite colony formation.
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