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Recombinant DNA (plasmids)

Page history last edited by Charles Forstbauer 14 years ago

 

Test is done. Page is closed.

Totaled 3/7/10 Mr F


 

 

 

 

This diagram does a great job showing how human insulin is made, and it is easy to understand. 

 

This image does a good job demonstrating how the sticky ends connect to each other.

 

 

 

 

I found this diagram very helpful because it is very detailed 

 

 

 chimericDNA.gif

Genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.

 

 

Above is a great diagram showing how Recombinant DNA is created. The gene and plasmid are spliced by the same nonrestrictive enzyme that leaves the same sticky ends on both. Then, the scrap of DNA from the gene is combined with the open "hole" in the plasmid. This creates recombinant DNA.

In the diagram above, a recombinant DNA is inserted into a bacteria. This bacteria produces a protein which is based onthe recombinant DNA; this is the recombinant protein.

 

13.2 Manipulating DNA

 

 

·         Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules. Different techniques are used to extract DNA from cells, to cut DNA into smaller pieces, to identify the sequence of bases in a DNA molecule, and to make unlimited copies of DNA.

·         Knowing the sequence of an organism’s DNA allows researchers to study specific genes, to compare them with the genes of other organisms, and to try to discover the functions of different genes and gene combinations.

·         genetic engineering- process of making changes in  the DNA code of living organisms

·         restriction enzyme- enzyme that cuts DNA at a specific sequence of nucleotides

·         gel electrophoresis- procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

·         recombinant DNA- DNA produced by combining DNA from different sources

·         polymerase chain reaction (PCR)- technique that allows molecular biologists to make many copies of a particular gene

 

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.>This video shows the process of a polymerase chain reaction. Instead of cloning, this process shows how a desired trait is cloned in another organism. 

 

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This video explains the history of engineering insulin, which is aided by the use of plasmids. The DNA is manipulated to create insulin, and as the man explains in the video, the study of changing DNA to make insulin started long ago.  

 

A long, but informative lecture from Stanford Professor Edward S. Mocarski, Jr.  http://www.youtube.com/watch?v=43QMu3LrE18  He does a very good job of clearly explaining the process of recombining DNA and how the DNA is manipulated. 

 

 

13.3 Cell Transformation

 

 

 

 

·         During transformation, a cell takes in DNA from outside the cell. This external DNA becomes a part of the cell’s DNA

 

·         If transformation is successful, the recombinant DNA is integrated into one of the chromosomes of the cell

·         plasmid- circular DNA molecule found in bacteria

·         genetic marker-gene that makes it possible to distinguish bacteria that carry a plasmid with foreign DNA from those that don’t

 

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>>This shows how restriction enzymes are used to create recombinant DNA.

 

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>>This video shows the cloning process within the organism and its DNA sequences. It is a helpful animation

 

What is a transgenic organism?

A transgenic organism is an organism that contains functional recombinant DNA.

 

What is a vector?

A vector is anything you use to deliver something. It is the "General term for a carrier by which a foreign DNA fragment can be transferred into the host cell." Basically, it is the host plasmid which delivers the recombinant DNA (made of the DNA and plasmid itself) into the plasmid containing bacterium.

 

 

 

 

Synthetic insulin production using recombinant DNA

One breakthrough in recombinant DNA technology was the manufacture of biosynthetic "human" insulin, which was the first medicine made via recombinant DNA technology ever to be approved by the FDA. Insulin was the ideal candidate because it is a relatively simple protein and was therefore relatively easy to copy, as well as being extensively used to the extent that if researchers could prove that biosynthetic "human" insulin was safe and effective, the technology would be accepted as such, and would open opportunities for other products to be made in this fashion.

The specific gene sequence, or oligonucleotide, that codes for insulin production in humans was introduced to a sample colony of E. coli (the bacteria found in the human intestine). Only about 1 out of 106 bacteria picks up the sequence. However, because the lifecycle is only about 30 minutes for E. coli, this limitation is not problematic, and in a 24-hour period, there may be billions of E. coli that are coded with the DNA sequences needed to induce insulin production.[6]

However, a sampling of initial reaction showed that Humulin was greeted more as a technological rather than a medical breakthrough, and that this sentiment was building even before the drug reached pharmacies.

The Economist concluded: "The first bug-built drug for human use may turn out to be a commercial flop. But the way has now been cleared-and remarkably quickly, too—for biotechnologists with interesting new products to clear the regulatory hurdles and run away with the prizes."[7]

Ultimately, widespread consumer adoption of biosynthetic "human" insulin did not occur until the manufacturers removed highly-purified animal insulin from the market. 

 

Conjugation

Conjugation is when two bacterial cells, one recipient and one donor, transfer a piece of genetic information from one to the other, as shown in the diagram below. Usually, plasmids are swapped/transfered from one bacterial cell to another, so that both have the same piece of genetic information.

 

     Plasmids are small, circular DNA that are found in bacteria.  They are usually only a few thousand base pairs long, and have a few genes or sometimes even one.  They are usually used for antibiotic purposes.

 

     The plasmids have to be altered in order to serve antibiotic purposes.  The plasmids are normally replicated at the same rate as chromosomes and bacteria can contain one or over 50 plasmids.  In order to alter them, the plasmid needs to be cut using restriction enzymes.  These are used to create sticky ends so

 

that a new gene could be put in and attached with ease.  The same enzyme must be used so that the cut is made in the same spot so that the base pairs from the new gene and the plasmid could match and close together.   

 

     There are two different types of restriction enzymes, that cut DNA in the middle of the restriciton endonuclease.  

 

          Type one- this recognizes specific sequences and cut DNA at a nonspecific site

 

          Type two- This is similar to the image above.  A palindromic sequence is recognized and a cut within the sequence is made.

 

     A Palindrome is anything that reads the same forwards and backwards.  Words such as racecar, and poop are examples of palindromes because they are read the same forwards and backwards.  In DNA the same concept is used.  One example of this is 

                                                                                                                                    5'- GAATTC -3'

                                                                                                                                    3'- CTTAAG -5'

 

     Why is all of this important?  It is important because this is a procces used for making things such as antibiotics.  There are proteins that are needed to make the antibiotics, but there needs to be a way of making the proteins.  So a gene is inserted into the plasmid of a bacteria, and the bacteria begins to make this protein.  Later as more bacteria are present, they are carying the gene and making more of the protein.  Than scientist could take the protein that they were making and use it for antibiotics. 

 

http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html

^ This website is very easy to understand how plasmids replicate.

 

http://www.youtube.com/watch?v=70lwiFIqejw&feature=related

^ This youtube video, is done by a student and is very well put together.  Since it is put together by students, it is easier for students to understand to the material.

 

Trouble With Plasmids

Many naturally occurring plasmids contain genes that provide some benefit to the host cell, fulfilling the plasmid’s portion of the symbiotic relationship. For example, some bacterial plasmids encode enzymes that inactivate antibiotics. Such drug-resistance plasmids have become a major problem in the treatment of a number of common bacterial pathogens. As antibiotic use became widespread, plasmids containing several drug-resistance genes evolved, making their host cells resistant to a variety of different antibiotics simultaneously. Many of these plasmids also contain “transfer genes” encoding proteins that can form a macromolecular tube, or pilus, through which a copy of the plasmid can be transferred to other host cells of the same or related bacterial species. Such transfer can result in the rapid spread of drug-resistance plasmids, expanding the number of antibiotic-resistant bacteria in an environment such as a hospital. Coping with the spread of drug-resistance plasmids is an important challenge for modern medicine.

About Plasmids 

The plasmids most commonly used in recombinant DNA technology replicate in E. coli.Generally, these plasmids have been engineered to optimize their use as vectors in DNA cloning. For instance, to simplify working with plasmids, their length is reduced; many plasmid vectors are only ≈3kb in length, which is much shorter than in naturally occurring E. coli plasmids. (The circumference of plasmids usually is referred to as their “length,” even though plasmids are almost always circular DNA molecules.) Most plasmid vectors contain little more than the essential nucleotide sequences required for their use in DNA cloning: a replication origin, a drug-resistance gene, and a region in which exogenous DNA fragments can be inserted

 

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