Structural Biochemistry/DNA recombinant techniques/Plasmid

A plasmid is an accessory chromosomal DNA that is naturally present in bacteria. Some bacteria cells can have no plasmids or several copies of one. They can replicate independently of the host chromosome. Plasmids are circular and double stranded. They carry few genes and their size ranges from 1 to over 200 kilobase pairs. Some functions of their genes include: providing resistance to antibiotics, producing toxins and the breakdown of natural products. However, plasmids are not limited to bacteria; they are also present in some eukaryotes (e.g., circular, nuclear plasmids in Dictyostelium purpureum).

A plasmid is a circular, double stranded DNA that is usually found in bacteria (however it does occur in both eukarya and prokarya). It replicates on its own (without the help of chromosomal DNA)and are used frequently in recombinant DNA research in order to replicate genes of interest. Some plasmids can be implanted into a bacterial or animal chromosome in which it becomes a part of the cell's genome and then reveals the gene of interest as a phenotype. This is how much research is done today for gene identification.

Plasmids contain three components: an origin of replication, a polylinker to clone the gene of interest (called multiple cloning site where the restriction enzymes cleave), and an antibiotic resistance gene (selectable marker).



Plasmids are usually isolated before they are used in recombinant techniques. Alkaline lysis is the method of choice for isolating circular plasmid DNA. This process is quick and reliable. You first obtain the cell that has the plasmid of interest and lyse it with alkali. This step is then followed by extracting the plasmid. The cell fragments are precipitated by using SDS and potassium acetate. This is spun down, and the pellet (cell/cell fragments) is removed. Next, the plasmid DNA is precipitated from the supernatant with the use of isopropanol. The plasmid is then suspended in buffer. Akaline lysis can give you different amounts of plasmid depending if it's a mini-, midi-, or maxi- prep.

Plasmids can be related to viruses because they can be independent life-forms due to their ability to self-replicate inside their host. Though they may be viewed as independent life-forms, they have a sense of dependency on their host. A plasmid and its host tend to have a symbiotic relationship. Plasmids can give their hosts needed packages of DNA carrying genes that can lead to mutual survival during tough times. Providing its host with such genetic information, plasmid allows the host to survive and at the same time allows itself to continue living in the host for generations.



Plasmids are used as vectors to clone DNA in bacteria. One example of a plasmid used for DNA cloning is called pBR322 Plasmid. The pBR322 plasmid contains a gene that allow the bacteria to be resistant to the antibiotics tetracycline and ampicillin. To use pBR322 plasmid to clone a gene, a restriction endonuclease first cleaves the plasmid at a restriction site. pBR322 plasmid contains three restriction sites: PstI, SalI and ecoRI. The first two restriction sites are located within the gene that codes for ampicillin and tetracycline resistance, respectively. Cleaving at either restriction site will inactivate their respective genes and antibiotic resistance. The target DNA is cleaved with a restriction endonuclease at the same restriction site. The target DNA is then annealed to the plasmid using DNA ligase. After the target DNA is incorporated into the plasmid, the host cell is grown in a environment containing ampicillin or tetracycline, depending on which gene was left active. Many copies of the target DNA is created once the host is able to replicate.

Another plasmid used as a vector to clone DNA is called pUC18 plasmid. This plasmid contains a gene that makes the host cell ampicillin resistant. It also contains a gene that allows it produce beta-galactosidase, which is an enzyme degrades certain sugars. The enzyme produces a blue pigment when exposed to a specific substrate analog. This allows the host to be readily identified. The gene for beta-galactosidase contains a polylinker region that contains several restriction sites. The pUC18 plasmid can be cleaved by several different restriction endonucleases which provide more versatility. When the polylinker sequence is cleaved and the target DNA is introduced and ligased, this inactivates the gene that codes for beta-galactosidase and the enzyme will not be produced. The host cell will not produce a blue pigment when exposed to the substrate analog. This allows the recombinant cells to be readily identified and isolated.

Cloning
Cloning is a method of recombining genes in order to take advantage of a bacteria's native ability to recreate plasmids. Engineered plasmids can be used to clone genetic material of up to 10,000 base pairs, the amount of genetic material is limited by the size of the plasmid. Because of the repetition of expressive genes within bacterial plasmids, it is possible to remove repeated genetic materials of the plasmid and replace it with desired traits. Most pre-engineered plasmids procured for laboratory use already contain an antibiotic resistance gene, polylinker site, and an origin of replication. The polylinker site is engineered to allow multiple unique cleaving sites that will allow needed DNA fragmentation. The origin of replication will mimic the genetic material of the bacteria that will be used for cloning.

Once the plasmid is acquired, the polylinker will be cleaved at two sites using specific endonucleases. Afterwards, the wanted DNA will also be cleaved from a different source with a different endonuclease. The cleaved DNA is sometimes amplified with a polymerase chain reaction. The desired DNA trait will be inserted into the now empty polylinker site. This replacement of the polylinker site with desired genetic traits is termed a cassette mutagenesis. The newly created plasmid will be mixed with bacteria, which will then be heat shocked or electric shocked to aide in the ability for the plasmid to act as a vector. After allowing the bacteria to reproduce, the antibiotic for which the engineered plasmid conferred resistance will be delivered. All still living bacteria will have acquired the desired traits of both the inserted DNA and the antibiotic immunity. The new proteins or biochemical structures from the inserted DNA can be gathered through different means.



Gene Mutations Using Plasmids
Deletions occur when one or more base pairs are removed from the DNA sequence. A large portion of DNA can be removed from the plasmid by using different restriction endonucleases to cut out a certain segment followed by ligation using DNA ligase to reform a new, smaller plasmid. A single or few base pairs can be removed by using multiple restriction endounucleases that cut near the sticky ends, followed by ligation.

Substitutions are a result of the change of a single amino acid in a protein sequence. This is typically accomplished by changing one (a point mutation) or more base pairs on the genetic code sequence in order to alter the amino acid at a particular site and is known as oligonucleotide-directed mutagenesis. An oligionucleotide is designed such that there is a one base pair difference at a particular site and this one base pair different will encode for a new residue. This oligionucleotide is annealed to the plasmid, which acts as the DNA template, and replication using DNA polymerase results in strands that contain this mutation. One stand of the replicated double helical DNA will be the parent chain and contain the original (wildtype) base sequence while the other chain will contain the new (mutant) strand of DNA that encodes for the new desired protein. By expressing the mutant chain, the desired protein can be harvested.

Insertions occur when an entire segment of DNA is introduced to a plasmid. The segment of DNA is known as a cassette and the technique is termed cassette mutagenesis. Plasmids are cut with restriction enzymes, removing a portion of DNA. Then specifically synthesized or harvested DNA is ligated into that region and the plasmid is expressed and studied.

It is also possible to create entirely new proteins and genes by joining together genes that are otherwise unrelated.

Types of Plasmids
Modes of Classification

Plamids are not required by their host cell for survival. They carry genes that provide a selective advantage to their host, such as resistance to naturally made antibiotics carried by other organisms. Antibiotic resistant genes produced by a plasmid will allow the host bacteria to grow in the presence of competing bacteria that produce these antibiotics. One way to classify plasmids is based on their ability to transfer to additional bacteria. Conjugative plasmids retain tra-genes, which carryout the intricate process of conjugation, the transfer of a plasmid to another bacterium. Conversely, non-conjugative plasmids are incapable of commencing conjugation, which consequently can only be transferred via conjugative plasmids. A transitional class of plasmids are considered to be mobilizable, contain only a subset of the genes necessry for a successful transfer. They have the ability to parasitize a conjugative plasmid by transferring at a high frequency exclusively in the presence of the plasmid. Currently, plasmids are used to manipulate DNA and could potentially be used as devices for curing disease. Figure 1-1: Illustrates the process of bacterial conjugation. It is possible for various plasmids to coexist in a single cell. A maximum of seven different plasmids have been found to coexist in a single E. coli. It is also possible to find incompatible related plasmids, where only one of the plasmids survive in the cell environment, due to the regulation of important plasmid functions. Hence, plasmids can be designated into groups according to compatibility.

Classification of Plasmids by Function Another approach to classify plasmids is according to their function.

There is a total of five major sub-groups:

Fertility Plasmids (F-Plasmids)- carry the fertility genes (tra-genes) for conjugation, the transfer of genetic information between two cells. F plasmids are also known as episomes because, they integrate into the host chromosome and promote the transfer of chromosomal DNA bacterial cells.

Figure 1-2: Illustrates a fertility plasmid.

Resistance Plasmids (R-Plasmids)- contain genes that encode resistance to antibiotics or poisons. Examples of R-plasmids found in Chapter 5 of "Biochemistry" by Berg include the following:

pBR322 Plasmid pBR322 was one of the first plasmids used for the purpose of cloning. It contains genes for the resistance to tetracycline and ampicillin. Insertion of the DNA at specific restriction sites can inactivate the gene for tetracycline (an effect known as an insertional inactivation) or ampicillin resistance.

Figure 1-3: Illustrates the pBR322 R-plasmid

pUC18 Plasmid pUC18/pUC19 has a greater versatility compared to pBR322. Comparable to pBR322, the pUC18 plasmid has an origin of replication and a selectable marker based on ampicillin resistance. Furthermore, this plasmid also contains a gene for beta-galactosidase, an enzyme that degrades certain sugars. while in the presence of a specific substrate analog, this enzyme produces a blue pigment that can be easily detected. Also, this enzyme has been equipped so that it has a polylinker region where many different restriction enzymes or combinations of enzymes can be used to cleave at different locations. Creating a greater variety in the DNA fragments that can be cloned. Interestingly, the insertion of a DNA fragment inactivates the beta-galactosidase. Thus if the blue pigment is not generated, it would be an indication that the DNA fragment was not inserted properly. pUC18 is similar to pUC19, but the MCS region is reversed.

Figure 1-4: Illustrates the pUC19 R-plasmid

Tumor Inducing Plasmids (Ti-Plasmids "Virulence Plasmids")- contain A. tumefaciens, which carry instructions for bacteria to become a pathogen by switching to the tumor state and synthesize opines, toxins and other virulence factors. The plasmid effectively transfers foreign genes into certain plant cells. Ti-Plasmids can can also be found in Chapter 5 of "Biochemistry" by Berg.

Figure 1-5: Illustrates the Ti-Plasmid

Degradative Plasmids- (Catabolic Plasmid) a type of plasmid that allows the host bacterium to metabolize normally ddifficult or unusual organic compounds such as pesticides.

Col- Plasmids- contain genes that encode for the antibacterial polypeptides called bacteriocins, a protein that kills other strains of bacteria. The col proteins of E. coli are encoded by proteins such as Col E1.

It is possible for a plasmid to belong to more than one of the above subgroups of plasmids.

Those plasmids that exist as only one or a few copies in a bacterium run the risk of being lost to one of the segragating bacteria during cell division. Those single copy plasmids implement systems which actively attempt to distribute a copy to both daughter cells.

Some plasmids include an addiction system, such as a host killing (hok) system of plasmid R1 in E. coli. Producing both a long lived poison and a short lived antidote. Those daughter cells that maintain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth rate because of the loitering poison from the parent cell.

Plasmids also replicate autonomously. Each plasmids contains its own origin sequence for DNA replication, but only a few of the genes needed for replication. Some plasmids come equipped with self-preservation genes, and they are called addiction modules. The addiction modules also force the host cell either keep the plasmid or die.

References for Types of Plasmids
Berg, Jeremy M., et al. "Biochemistry". 6th ed. W.H. Freeman and Company, NY, 2007.

Uses, Applications, and Significance
Plasmid provides a versatile tool in genetic engineering because of its unique properties as a vector. Plasmids are utilized to create transgenic organisms by introducing new genes into recipient cells. For example, the Ti plasmid from the soil bacterium Agrobacterium tumefaciens is very valuable in plant pathology in developing plants with resistance to diseases such as holcus spot on leaves and crown gall tumors.

Plasmid also carries medical significance because of its role in antibiotic synthesis. Streptomyces coelicolor plasmid can give rise to thousands of antibiotics, as well as that of S. lividans or S. reticuli. In another example, E. coli plasmids are used to clone the gene of penicillin G acylase, the enzyme that turns penicillin G into the antibacterial 6-amino-penicillanic acid. Once again, these cloning processes are carried out with the assistance of type II restriction enzyme to put the gene of interest into the plasmid vector.

In DNA recombinant technology, plasmid-based reporter gene are crucial as they allow observation of organisms in real time. The gene for Green Fluorescent Protein can be integrated into a plasmid of the organism under investigation. The encoded protein is small and does not alter the function of the host protein. This feature of GFP makes it very easy to observe cell dynamics.

These are only a few among many techniques, applications and uses of plasmids developed throughout the years. The future of plasmid engineering looks very promising with many more examples and opportunities to come.

Alkaline Lysis Mini Plasmid Prep
In order to purify the plasmid DNA from the bacterial cell, alkaline lysis plasmid prep will need to be done. Three solutions will be prepared as following:

Solution1: 50mM glucose, 25mM Tris-HCL pH8, 10mM EDTA, 50 microgram/ml RNase

The purpose of adding glucose is to increase the solute concentration outside the cell, so once the cells are broken open, the osmotic pressure will draw water out of the cell and carry the plasmid with it. EDTA is a chelator of divalent cations such as Mg2+, Ca2+, and Mn2+. These cations will be bind away and remove from the solution. Mg2+ is the cofactor of DNase, so if it is bind away then DNase will not be able to digest DNA hence DNA will remain intact. RNase A will degrade any bacterial RNA present.

Solution2: 0.2M NaOH, 1%SDS 

SDS lyses cells by dissolving the cell membrane. The high pH created by the NaOH will denature the bacterial proteins so they will precipitate out of solution. The extreme pH will denature DNA by separate two strands of the double helix giving single strand of bacterial chromosome and plasmid DNA. However, the small interlocked plasmid strands will stay together because it is bind tightly hence NaOH cannot get in.

Solution3: 3M Kacetate( 29.4g KOAc + 11.5 ml glacial acetic acid/ 100ml), pH 5.5 

The acetate buffer will bring the pH of the solution to a more neutral pH. This will allow DNA strands to re-anneal. The chromosomal DNA is too complex and big to re-anneal under these condition hence only the plasmid DNA re-anneal. The K+ ions will bind with SDS (-) forming KDS which will precipitate out from the solution.