Molecule | Turkish Chemistry
May 17

Plasmids

A plasmid is an independent, circular, self-replicating that carries only a few genes. The number of plasmids in a generally remains constant from generation to generation. Plasmids are autonomous and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using (see also alkaline lysis)

They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid is the basis of recombinant technology. Plasmids

A plasmid is an independent, circular, self-replicating that carries only a few genes. The number of plasmids in a generally remains constant from generation to generation. Plasmids are autonomous and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using (see also alkaline lysis) They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid is the basis of recombinant technology.

There are two categories of plasmids. Stringent plasmids replicate only when the chromosome replicates. This is good if you are working with a that is lethal to the . Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to chromosome.

So how do we manipulate these plasmids?

    1. Mutate them using restriction enzymes, ligation enzymes, and PCR. Mutagenesis is easily accomplished by using restriction enzymes to cut out portions of one genome and insert them into a plasmid. PCR can also be used to facilitate mutagenesis. Plasmids are mapped out indicating the locations of their origins of replication and restriction enzyme sites.

    2. Select them using genetic markers. Some are antibiotic resistant. While this is a serious health problem, it is a godsend to molecular scientists. The gene that confers antibiotic resistance can be added (ligated) to the gene you are inserting into the plasmid. So every plasmid that contains your target gene will not be killed by antibiotics. After you transfect your bacterial cells with your engineered plasmid (the one with the target gene and the antibiotic resistant marker), you incubate them in a nutrient broth that also contains antibiotic (usually ampecillin). Any cells that were not transfected (this means they do not have your target gene in them) are killed by the antibiotic. The ones that do have the gene also have the antibiotic resistant gene, and therefore survive the selection process.

    3. Isolate them (such as with alkaline lysis)

    4. Transform them into cells where they become vectors to transport foreign genes into a recipient organism.

There are some minimum requirements for plasmids that are useful for recombination techniques:

    1. Origin of replication (ORI). They must be able to replicate themselves or they are of no practical use as a vector.

    2. Selectable marker. They must have a marker so you can select for cells that have your plasmids.

    3. Restriction enzyme sites in non-essential regions. You don’t want to be cutting your plasmid in necessary regions such as the ORI.

In addition to these necessary requirements, there are some factors that make plasmids either more useful or easier to work with.

    1. Small. If they are small, they are easier to isolate (you get more), handle (less shearing), and transform.

    2. Multiple restriction enzyme sites. More sites give you greater flexibility in cloning, perhaps even allowing for directional cloning.

    3. Multiple ORIs. It is important to note that two genes must have different ORIs if they are going to be inserted in the same plasmid.

 

May 17

RIP (radio-immune precipitation)

RIP (radio-immune precipitation)

Radio-immune precipitation (RIP) can take over where a Western blot will fail you. Western blots let you know how much protein has accumulated in a sample. If you are more interested in the rate of in a cell, or if your protein degrades too quickly to be detected by a Western blot, then RIP is definitely a technique you’ll want to know about. RIP also detects protein-protein interaction, while Western blotting can’t.

This chart should help illustrate the differences between RIP and Western blotting.

 

Let’s look at this technique in greater detail.

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    • [35S] labels methionine—high energy, high specificity
    • [3H] labels amino acids—low energy
    • [14] labels amino acids—low specific activity 

    • 1. Begin by radioactively labeling your cells.

    You have the following choices:

     

    2. Extract-release your protein mixture. You’ll need to make antibodies against one protein, then incubate your protein in them. This will give you an antibody-protein complex, but it will also leave you with some free antibodies floating around. So you’ll need to purify your antibody-protein complexes.

    3. Use the bacteria staphlococcus aureus in excess to purify your protein. Before incubating your protein in , fix it with formaldehyde or glutaraldehyde to kill the , since it’s a pathogen. The formaldehyde or glutaraldehyde will crosslink all of the proteins in the cell. has on its surface a protein that binds to the tail of antibodies (to the Fc portion of the Ig molecule). That means it binds both free antibodies and your radiolabeled antibody-protein complexes.

    4. Centrifuge the solution to get a precipitate. Discard the solution and save the pellet. Elute the antibody-protein complexes by boiling them in SDS sample buffer. This denatures the proteins and removes the antibodies from the and the radiolabled proteins.

     5. Run this on an SDS gel. Perform an autoradiograph and develop the x-ray film. You’ll see a dark spot on the film for whatever protein was bound by the antibody.

     

May 17

PCR (polymerase chain reaction)

Let’s say you have a biological sample with trace amounts of in it. You want to work with the , perhaps characterize it by sequencing, but there isn’t much to work with. This is where PCR comes in. PCR is the amplification of a small amount of into a larger amount. It is quick, easy, and automated. Larger amounts of mean more accurate and reliable results for your later techniques.

The techniques was developed by Nobel laureate biochemist Kary Mullis in 1984 and is based on the discovery of the biological activity at high temperatures of polymerases found in thermophiles (bacteria that live in hot springs).Most polymerases (enzymes that make new ) work only at low temperatures. But at low temperatures, is tightly coiled, so the polymerases don’t stand much of a chance of getting at most parts of the .

But these thermophile polymerases work at 100C, a temperature at which is denatured (in linear form). This thermophilic polymerase is called Taq polymerase, named after Thermus aquaticus, the bacteria it is derived from.

Taq polymerase, however, has no proofreading ability. Other thermally stable polymerases, such as Vent and Pfu, have been discovered to both work for PCR and to proofread.

You’ll need four things to perform PCR on a sample:

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      1. The target sample. This is the biological sample you want to amplify from.

      2. A . Short strands of that adhere to the target segment. They identify the portion of to be multiplied and provide a starting place for replication.

      3. Taq polymerase. This is the enzyme that is in charge of replicating . This is the polymerase part of the name polymerase chain reaction.

      4. Nucleotides. You’ll need to add nucleotides (dNTPs) so the polymerase has building blocks to work with.

There are three major steps to PCR and they are repeated over and over again, usually 25 to 75 times. This is where the automation is most appreciated.

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          1. Annealing temperature. Starts at the low end of what you think will work, then move up as necessary. If the temperature is too low, the primers will make more mistakes and you’ll get too many bands when you run your sample on a gel. If the temperature is too high you will get no results and your gel will be blank. You want to be about 3C to 5C below the melting temperature (Tm). A rough formula for determining Tm is Tm=4(G + C) + 2(A + T).

          2. Magnesium concentration. You want your Mg2+ concentration to be about 1.5mM to 3mM. If you go too high, the polymerase will make more mistakes.

          3. Think carefully about design. Both primers should have approximately the same Tm so they both anneal at the same temperature. Two out of three bases on the 3′ end should b G or C to get good hybridization (G and C have three H-bonds so you get better polymerization). Lastly, avoid dimers, which occur when the primers have ends that will anneal to each other. This will produce NO product.

          4. More is not necessarily better. More polymerase produces more nonspecific product, so don’t just carelessly dump in a bunch of polymerase. Additionally, PCR reactions don’t work if there is too much .

      • 1. Your target sample is heated. This denatures the , unwinding it and breaking the bonds that hold together the two strands of the molecule, leaving you with single stranded (ssDNA).

      2. Temperature is reduced and the is added. The now have the opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of to be amplified and provides a starting place for replication.

      3. New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new pieces of ssDNA that are complimentary to the portions marked by the primers. The job of Taq polymerase is to move along the strand of and use it as a template for assembling a new stand that is complimentary to the template. This is the chain reaction in the name polymerase chain reaction.

      PCR is so efficient because it multiplies the exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of that is made can serve as a template for new ones.

      Perhaps the most important thing to remember is to be very aware of contamination. If, for example, you unknowingly slough off a piece of skin into your sample, then your may be amplified in the PCR reaction.  Here are some other factors to optimize your results with PCR:

      RT-PCR

      Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA . The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA . After you add your RNA sample to the PCR machine, add a as usual and allow it to anneal to your target molecule. Then add RT along with dNTPs, which will elongate the and make a cDNA copy of the RNA and run the PRC reaction as usual. The product of RT-PCR is a double stranded molecule analogous to the target segment of the RNA molecule.

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