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Shannon Greenfelder
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∙ 15y agoto make bacteria take up a plasmid, you need to add Ca+2 into solution to make the membrane porous. also to select for the bacteria that has the plasmid you need to have something like an ampicilin resistance sequence in there as well and grow the culture in an amp culture
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In the process of human gene cloning using recombinant plasmids what is the bacterial plasmid?
functions as a vector
What are the genes on the pVIB plasmid?
It contains a gene for luciferase, a Lux gene (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). This causes bacterial cells to glow!
Describe the use of plasmids as vectors in biotechnology?
Plasmids are often used as expression vectors in biotechnology. Plasmids are small, circular or linear pieces of DNA containing non-essential genes that are found in all life, although much more common in prokaryotes, especially bacteria. These genes confer abilities such as metabolizing a previously unusable compound, building an amino acid previously unbuildable, or even antibiotic resistance. Plasmids are used in research to induce the expression of a gene usually not found in the given organism. For example, you can construct a plasmid with a bacterial promoter connected to the gene for a human protein through a process called 'cloning'. The plasmid with the human gene can then be introduced into bacteria by transforming a competent gram-negative with the plasmid. Usually the plasmid also has an antibiotic resistance gene in addition to the target gene. This antibiotic resistance can be used to select for bacteria containing the plasmid. For example, the most common resistance gene is ampicillin resistance gene. If you grow the transformed bacteria in a culture containing ampicillin, only bacteria containing the antibiotic resistance, and therefore containing the plasmid, can survive. This will ensure that what you have is a pure culture of bacteria containing the plasmid. After selection, these bacteria can be cultured in suitable media to increase their numbers to a point that their production of the human protein becomes appreciable. Then these bacteria are usually lysed (killed) to extract the protein. Sometimes, however, these bacteria can also be made to secrete the protein into the medium.
Which enzyme would cut the human DNA shown in Part A on both sides of the vgp gene but not inside the gene?
1. Which enzyme(s) would cut the human DNA shown in Part A on both sides of the vgp gene, but not inside the gene? Answer: BamHI, HaeIII, and HindIII 2. Which enzymes(s) would cut the plasmid without disrupting the function of the amp^R gene? Answer: BamHI, EcoRI, and HaeIII 3. Which enzyme(s) would produce sticky ends when cutting both the human DNA and the plasmid? Answer: BamHI, EcoRI, and HindIII 4. Which one restriction enzyme satisfies all three of the requirements listed above? Answer: BamHI only
Bacteria containing a plasmid into which the eukaryotic gene has integrated would grow where?
the ampicillin broth and the nutrient broth
A plasmid that contains a gene for human growth hormone is and example of what?
recombinant dna
What is the term for a plasmid that contains a foreign gene?
Recombiant DNA
What is an extra loop of DNA that contains antibiotic resistance what gene?
Plasmid
When is a plasmid considered a recombinant plasmid?
When the original function of the gene in the plasmid is altered or another gene is inserted in the non- coding region of the plasmid is called the recombinant plasmid.
Why does bacteria that contains plasmid glow in uv light?
the plasmid contains a certain gene, which codes for the "Green Flourescent Protein." So you put the plasmid in the bacteria, the plasmid starts making that protein in the bacteria, and boom you've got glowing bacteria. works for bunnies and monkeys too, apparently =)
How does a human insulin genes become part of a plasmid?
1. Scientists remove plasmids, small rings of DNA, from bacterial cells. 2. An enzyme cuts open the plasmid DNA. The same enzyme removes the human insulin gene from its chromosome. 3. The human insulin gene attaches the open ends of the plasmid to form a closed ring. 4. Some bacterial cells take up the plasmids that have the insulin gene. 5. When cells reproduce, the news cells will contain copies of the engineered plasmid. The foreign gene directs the cell to produce human insulin.
In the process of human gene cloning using recombinant plasmids what is the bacterial plasmid?
functions as a vector
Why does a gene fit into the opening in the plasmid?
When genes transfer the tra gene nicks the DNA at its origin of transfer creating a pilus so the chromosome (which contains the gene) can transfer to the other DNA.
How do you express a human gene in E. coli?
E.coli is used to express human genes because it can be easily grown in the lab. The gene is extracted from the DNA (by doing a partial digest with a restriction enzyme), and given a cohesive sticky end with a linker or adapter. It is then ligated to a plasmid vector, which had a restriction site compatible with the ends on the gene, eg if the plasmid contains a BamH1 site then you would add a linker or adapter which is compatible with the 5'GGATCC3' BamH1 recognition sequence. The cells are transformed (made to take up the plasmid vector) by chemical treatment; they are mixed with the plasmid, then a strong concentration of calcium (Ca2+) ions is added to the mixture to make the E.coli's membranes porous. The mixture is then heated to heat-shock the cells, to approx 50 degrees C for one minute. They are then cooled and allowed to recover in a nutrient rich broth at optimum temperature. This is a very inefficient process - only about 1 cell in every million is transformed. The pUC18 plasmid vector is useful because it contains the gene for ampicillin resistance. Any cells which subsequently grow on a medium containing ampicillin, therefore, have been transformed with the plasmid vector. It is also useful because it contains a beta-galactosidase gene, which itself contains the recognition site for a number of restriction enzymes, including BamH1. This is good because you can tell if the vector has taken up the gene you are trying to express when the vector no longer codes for the beta-galactosidase protein product. If the vector has been ligated with the gene, the gene will have disrupted the beta-galactosidase gene. This can be tested with IPTG (an auto-inducer) and X-gal, which will turn colonies of E.coli with the beta-galactosidase gene intact blue (ie, those without the gene of interest). Colonies which have had their beta-galactosidase gene destroyed by the ligation of the gene of interest will be colourless in the presence of X-gal and IPTG. These colonies are all clonal, so all cells in colourless colonies contain copies of the pUC18 plasmid vector which has been ligated with the human gene.
Can genetic engineering techniques treat cystic fibrosis?
Yes, there are two similar techniques in which i am aware of.AdenovirusesFirstly adenoviruses are made harmless by interefering with a gene involved in replication. A healthy from of the CFTR gene is extracted and cut with restriction endonucleases, the same enzyme is used to cut a bacterial plasmid. The gene and plasmid are mixed together along with DNA ligase to anneal the phosphosugar framework of the DNA fragment and bacterial plasmid. The plasmid is then mixed with epithelial cells. The plasmid is then isolated and purified and places into adenoviruses. These are then placed onto the nostrils of individuals with cystic fibrosis. The viruses find their way to epithelial cells in the airways and injected their DNA. The DNA contains the functional CFTR gene, the cells can then produce fucntional CFTR proteins.LiposomesA healthy gene is extracted from a human. This gene is then inserted into a bacterial plasmid, in a similar manner as discussed above. The bacterial plasmids are then inserted into bacteria. These are allowed to grow and divide, producing large quantities of the plasmid, with the required gene. These plasmids are then extracted and coated in a lipid soluble substance. They are then put into nasal sprays and sprayed onto the nostrils of effected individuals.
What type of gene is used to distinguish bacteria that carry a plasmid containing foreign DNA from those that don' t?
The plasmid that contains foreign DNA is engineered to also carry an antibiotic resistance gene. This antibiotic resistance gene codes for a protein that is able to inactivate an antibiotic thus keeping the cell alive. In the absence of the antibiotic resistance gene, the cells would not survive when exposed to an antibiotic. After transfection (the process of inserting the plasmid carrying the foreign gene into cells), the cells are gown in media containing an antibiotic. Cells that contain the plasmid (and therefore contain the antibiotic resistance gene) are able to survive in this medium. Cells that do not contain the plasmid (and therefore lack the antibiotic resistance gene) do not survive in this medium. The process described above is called selection
What would be the advantages of inserting into a plasmid gene with a highly visible phenotype?
Inserting a plasmid gene into the organism gives us three situation that one is the foreign cell may not pick up the plasmid the second chance is it is picked up may not expressed and in the third case it is expressed and therefore you can have the gene of interest. This is the one main advantage of studying the gene of interest by inserting a plasmid gene.
