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Recently I was studying Biotechnology. When I went through the texts, I had a doubt: both plasmids and gene of interest are made of DNA stretches and bacteria directly absorb plasmids in a test tube (where reactions take place). Why is the gene of interest not directly put in the test tube instead of recombinant DNA Technology (using Edited plasmids), so that it gets absorbed by bacteria?
A plasmid has a site necessary for replication of genetic material know as the origin of replication (ori) since gene of interest (GOI) is just a segment of DNA it only posseses the sequence of interest and lacks an ori site, due to this it will not be able to replicate by itself. Replication of GOI is important for product formation in r-DNA technology (mostly protien) as when the host cells divides the GOI is duplicated and is transferred to both daughter cells. This way the number of recombinant cells will increase hence producing more of the final product.
Genetic Engineering: DNA Technology Applications
The use of recombinant DNA technology has become commonplace as new products from genetically altered plants, animals, and microbes have become available for human use. In 1997, Dolly made headlines as the first successfully cloned large mammal (sheep). Since then there have been many similar advances in medicine, such as treatments for cancer many advances in agriculture, such as transgenic insect-resistant crops and many advances in animal husbandry, such as growth hormones and transgenic animals (an animal that has received recombinant DNA).
Most biotechnologists envision DNA technological applications as one of the new frontiers in science with tremendous growth and discovery potential.
Genetic engineering has resulted in a series of medical products. The first two commercially prepared products from recombinant DNA technology were insulin and human growth hormone, both of which were cultured in the E. coli bacteria. Since then a plethora of products have appeared on the market, including the following abbreviated list, all made in E. coli:
A vaccine is usually a harmless version of a bacterium or virus that is injected into an organism to activate the immune system to attack and destroy similar substances in the future.
- Tumor necrosis factor. Treatment for certain tumor cells
- Interleukin-2 (IL-2). Cancer treatment, immune deficiency, and HIV infection treatment
- Prourokinase. Treatment for heart attacks
- Taxol. Treatment for ovarian cancer
- Interferon. Treatment for cancer and viral infections
In addition, a number of vaccines are now commercially prepared from recombinant hosts. At one time vaccines were made by denaturing the disease and then injecting it into humans with the hope that it would activate their immune system to fight future intrusions by that invader. Unfortunately, the patient sometimes still ended up with the disease.
With DNA technology, only the identifiable outside shell of the microorganism is needed, copied, and injected into a harmless host to create the vaccine. This method is likely to be much safer because the actual disease-causing microbe is not transferred to the host. The immune system is activated by specific proteins on the surface of the microorganism -e. DNA technology takes that into account and only utilizes identifying surface features for the vaccine. Currently vaccines for the hepatitis B virus, herpes type 2 viruses, and malaria are in development for trial use in the near future.
Crop plants have been and continue to be the focus of biotechnology as efforts are made to improve yield and profitability by improving crop resistance to insects and certain herbicides and delaying ripening (for better transport and spoilage resistance). The creation of a transgenic plant, one that has received genes from another organism, proved more difficult than animals. Unlike animals, finding a vector for plants proved to be difficult until the isolation of the Ti plasmid, harvested from a tumor-inducing (Ti) bacteria found in the soil. The plasmid is ?shot? into a cell, where the plasmid readily attaches to the plant's DNA. Although successful in fruits and vegetables, the Ti plasmid has generated limited success in grain crops.
Creating a crop that is resistant to a specific herbicide proved to be a success because the herbicide eliminated weed competition from the crop plant. Researchers discovered herbicide-resistant bacteria, isolated the genes responsible for the condition, and ?shot? them into a crop plant, which then proved to be resistant to that herbicide. Similarly, insect-resistant plants are becoming available as researchers discover bacterial enzymes that destroy or immobilize unwanted herbivores, and others that increase nitrogen fixation in the soil for use by plants.
Geneticists are on the threshold of a major agricultural breakthrough. All plants need nitrogen to grow. In fact, nitrogen is one of the three most important nutrients a plant requires. Although the atmosphere is approximately 78 percent nitrogen, it is in a form that is unusable to plants. However, a naturally occurring rhizobium bacterium is found in the soil and converts atmospheric nitrogen into a form usable by plants. These nitrogen-fixing bacteria are also found naturally occurring in the legumes of certain plants such as soybeans and peanuts. Because they contain these unusual bacteria, they can grow in nitrogen-deficient soil that prohibits the growth of other crop plants. Researchers hope that by isolating these bacteria, they can identify the DNA segment that codes for nitrogen fixation, remove the segment, and insert it into the DNA of a profitable cash crop! In so doing, the new transgenic crop plants could live in new fringe territories, which are areas normally not suitable for their growth, and grow in current locations without the addition of costly fertilizers!
Neither the use of animal vaccines nor adding bovine growth hormones to cows to dramatically increase milk production can match the real excitement in animal husbandry: transgenic animals and clones.
Transgenic animals model advancements in DNA technology in their development. The mechanism for creating one can be described in three steps:
- Healthy egg cells are removed from a female of the host animal and fertilized in the laboratory.
- The desired gene from another species is identified, isolated, and cloned.
- The cloned genes are injected directly into the eggs, which are then surgically implanted in the host female, where the embryo undergoes a normal development process.
It is hoped that this process will provide a cheap and rapid means of generating desired enzymes, other proteins, and increased production of meat, wool, and other animal products through common, natural functions.
Ever since 1997 when Dolly was cloned, research and experimentation to clone useful livestock has continued unceasingly. The attractiveness of cloning is the knowledge that the offspring will be genetically identical to the parent as in asexual reproduction. Four steps describe the general process:
Many techniques in molecular biology are used to find out where a given gene is located within the human genome. This is not an easy task, since the human genome contains thousands of genes, many of which have not yet been found or sequenced. Of particular use in this task are DNA probes.
One way of finding a specific gene is through the use of a DNA probe, a relatively short single stranded DNA molecule that is complementary to a sequence on the gene of interest. In other words, if a segment of the gene of interest is known to be: AGTTCG, the complementary segment of the DNA probe would be TCAAGC, because A binds T and C binds G. (Actually, because of certain structural details about DNA molecules, the complementary sequence should actually be written as CGAACT, which is TCAAGC backwards. However, we are using the technically incorrect format here, to avoid confusion.)
An actual DNA probe would probably consist of at least a few dozen nucleotides complementary to a segment of the same length on the gene of interest. The probe is made to be radioactive, so that it can be detected easily.
Since a DNA probe will bind to single-stranded DNA, a technique called Southern Blotting can be performed which separates the double-stranded sample DNA into single strands and transfers them to a nylon membrane. When the probes are incubated with the membrane in a solution, they bind to complementary regions in the DNA and become "stuck" to the membrane. Afterwards, the membrane is put into contact with a sheet of photographic film that is sensitive to radioactive emission. Only those sections of the sample where the gene is located shows up as a dark spot on the paper, because these are the only sections bound to a radioactive probe.
DNA probes have several applications in molecular biology, including genetic methods of disease prediction and diagnosis , where probes that identify gene sequences known to be responsible for diseases are used. DNA probes can also be used in DNA fingerprinting , a technique often employed by forensic scientists to determine whether DNA found at a crime scene matches a suspect's DNA.
Using plasmids for cloning
One of the more common techniques available to scientists working in molecular biology is cloning. In this technique, sequences of DNA containing genes of interest are inserted into vectors which are then used to introduce these genes into cells or organisms to study the effects of the expression of the genes.
Vectors are based on bacterial plasmids – short circular pieces of DNA separate to the main bacterial chromosome which may be transferred between bacteria. Scientists source plasmid vectors from biological supply companies, which create them by ligating together pre-existing genes and sequences of DNA built from scratch using sequencing technology. A map of an example of a commercial vector (the pGEM-T Easy system) is presented in the figure below.
Note that this map shows a number of regions contained within the vector. The numbers refer to how many base pairs along the sequence (out of a total of 3015) a particular region is found.
In our experiment, the pGEM-T Easy vector has had a short fragment of the gene for the protein polo-like kinase 1 (PLK1) cloned in at the insertion site (located at around “3 o’clock” if you imagine the picture of the vector to be a clock face). This portion of the gene codes for a region of the protein called the polobox domain, which assists in the localization of the protein at various point during the cell cycle.
Prior to today’s project, this cloned vector was used to transform E. coli cells. The cells were used to inoculate an agar plate containing ampicillin and the plates incubated overnight. Because of the presence of the ampicillin resistance gene in the pGEM-T vector, the only cells to grow into colonies were those that had been transformed by the plasmid. These colonies were then used to inoculate a culture broth, which has been provided to you.
The alkaline lysis mini-plasmid preparation
Using transformed bacteria is a very efficient means to generate DNA needed for research. Bacteria have minimal requirements for nutrition and so the production of large quantities of DNA can be done quickly and at a minimal cost. Once a culture of transformed cells is established, this culture can be used to seed new cultures, so transformation need only be performed once. However once we have our culture, we need a way of recovering the DNA in a relatively pure form.
DNA (including plasmid DNA) is not generally secreted by cells. In order to recover it, we need to disrupt the cells and then purify the DNA we need from the other cellular contents. This is the purpose of the alkaline lysis mini-plasmid preparation (or mini-prep).
The first stage of the mini-prep involves bursting the cells using an alkaline solution. This releases their contents into the surrounding liquid. An acidic solution is then added, which neutralizes the alkaline solution and denatures the proteins, causing them to become insoluble. They can be removed from the cell lysate through centrifugation (see figure below).
The second stage of the mini-prep involves passing the cell lysate through a column. These columns bind onto plasmid DNA, and allow chromosomal DNA and other cell products to pass through. After washing the column several times, we can make the column release the plasmid DNA by passing through an elution solution (see figure below).
By the end of the mini-prep procedure, you should have approximately one drop of a colourless liquid. To demonstrate that you have recovered the plasmid DNA you will need to run the sample on an electrophoresis gel. However, before your sample is ready to run, you must first prepare it using a restriction digest.
Plasmid DNA is circular. For DNA to be demonstrated on a gel, it needs to be linearised. This is done by using enzymes to cut the DNA (think of cutting a rubber band once to obtain a straight strip of rubber). Restriction endonucleases are enzymes which cut the DNA strand at very specific locations, normally given by sequences of half a dozen or so base pairs called restriction sites. If we know the sequence of a length of DNA, we can select enzymes which cut the DNA once (ie. the restriction site sequence occurs once in the entire DNA sequence) or even twice. If a plasmid is cut twice, you should end up with DNA fragments of two different sizes. Molecular biologists often use this double cutting to “drop out” an insert they have cloned into a vector.
The pGEM-T Easy vector has been created with a number of restriction sites on either side of the insertion point. Some of these restriction sites are only found on one side of the insertion point, and so can be used to linearise the vector to make it ready for electrophoresis. Others are found on both sides, and so may be used to drop out the insert to check its size. In this exercise, we will be using the enzyme EcoRI which has restriction sites just upstream (before) and downstream (after) the insertion site. This will allow us to drop out the polobox insert, resulting in DNA of two different sizes : around 3015 base pairs long for the vector, and 800 base pairs long for the insert. The two linearised fragments are now ready to be demonstrated using electrophoresis.
Further information on restriction digests can be found here.
Agarose gel electrophoresis
If we wanted to sort sand from gravel from larger rocks, we would use a series of sieves of different sizes. Each sized sieve lets smaller particles pass through but retains the larger fragments. Electrophoresis can be thought of as a sieve for large molecules like DNA or protein.
In agarose gel electrophoresis, a DNA sample is loaded towards one end of a block of a jelly-like substance called agarose. When an electrical current is passed through the gel, the DNA molecules are pushed through the gel away from the negative electrode (DNA has an overall negative charge and like charges repel). Smaller fragments of DNA can move more easily through the gel than larger fragments, so in a given period of time, DNA of different sizes accumulates in regions of the gel. If we include a dye which binds to the DNA, , these regions are visible as bands – the further towards the positive electrode a band is located, the smaller the fragments of DNA are found in that band.
To get an idea of the size of a band seen on a gel, we always run a sample consisting of a mixture of DNA fragments of known sizes alongside our test samples. This is called a marker, or a “ladder”, as the multiple bands of DNA seen on the gel resembles the rungs on a ladder. By matching the position of a band in our test sample to those representing DNA of known size in the ladder, we can estimate the size of DNA fragments in our test. The part of the PLK1 gene which codes for the polobox domain is 800 base pairs (bp) long. Therefore, if our digest has been a success, we should see a band corresponding to our DNA markers which is 800 base pairs long representing the polo-box insert, and another representing the pGEM-T vector at 3000 base pairs long.