Gene+cloning

=Gene cloning=


 * Gene cloning ** is the process of inserting a gene of importance into a recipient organism, usually a bacterium, in order to replicate that gene many times and mass-produce a useful gene product. The recipient organism is called the **host**, and the material used to introduce the gene, usually a plasmid or bacteriophage, is called the **vector.** Ideal vectors have high rates of replication. Gene cloning is an important procedure in genetic engineering that allows us to produce a large amount of a useful gene product: for instance, insulin can be made for diabetic individuals by cloning genes from pancreatic cells.

 The procedure first involves the digestion of target DNA containing the gene of interest, and the vector which will contain the gene (usually a bacterial plasmid) with the same restriction enzyme in order for them to have 'sticky ends' (areas of unpaired bases) that are complementary to each other and can easily anneal by hydrogen bonding. The digested target gene and the digested plasmid are then mixed together with DNA ligase to secure the annealing by forming covalent phosphodiester bonds between the ends of the target gene and the ends of the plasmid. Once the plasmid has been engineered (it is now called a recombinant plasmid), it can be taken up by a bacterium in an environment of calcium chloride, which promotes DNA uptake by bacteria.

 Bacteria containing the recombinant plasmid are called transformants, and we can test to see which bacteria in a colony are transformants by also inserting **genetic markers ** into the plasmid. For instance, a gene for antibiotic resistance may be inserted into the plasmid containing the gene we want to clone. This means that transformants can be identified by placing bacteria in an antibiotic and seeing which bacteria are resistant (those which are resistant contain the gene of interest). Transformants are then cultured in suitable conditions in order for them to replicate and synthesise large amounts of useful gene produ ct.

Another genetic marker is implicit in plasmids where the start of the clone site runs through another gene. An example is pBluescript, a plasmid with a gene called //lac//Z that encodes beta-galactosidase and, when placed on a medium of colourless synthetic galactoside, produces an insoluble blue dye. The cloning site is within the //lacZ// gene so that, in a recombinant plasmid, the //lacZ// gene is disrupted and thereby inactivated. Thus recombinants can be distinguished by their inability to produce the blue dye on such a medium.

Another vector that may be used in gene cloning, besides a plasmid, is a bacteriophage virus that has had its lysogenic genes replaced by the gene of interest. Because such recombinant phages are only capable of the lytic cycle, this is a good genetic marker in itself; bacteria transformed by the recombinant phage will appear as lysed plaques on a petri dish, while non-transformed bacteria will lysogenise.


 * The next section of this article describes the procedure of gene cloning in depth, using a bacterial plasmid as a vector. **

Unlike the larger chromosomal DNA which is tethered to the membrane, bacterial plasmids are small and soluble, which makes them easy for extraction.
 * Isolating a plasmid **

First, a bacterial colony is grown in 3mL culture in a shaker overnight, at 37C. This can produce 10 9 - 10 10 bacteria and, since each bacteria contains plasmids on the order of 10 2, this means 10 11 - 10 12 plasmids are made available for extraction. To extract the plasmids, the cells are centrifuged into a pellet and the supernatant discarded. The cells are gently broken up to release only small and soluble components, including the plasmids but excluding the genomic DNA. Any RNA present is digested using the enzyme RNase, and proteins are removed by phenol extraction. The water phase, containing the DNA, floats on top of the phenol phase and can be easily removed. The DNA is then purified further by precipitation with alcohol and salt. Because DNA is hydrophilic, the alcohol solution (due to its hydrophobicity) causes the DNA molecules to aggregate into macroscopic complexes than can be isolated by centrifugation. Once centrifuged, the pellet is removed and dried and resuspended in a small volume of slightly alkaline buffer (pH 7.5-8), of 10mM Tris-HCl (pH8) and 0.1mM EDTA (pH8).

It is then possible to visualise the plasmid on a gel. From this point, it is also possible to cut the plasmid into fragments and customise it to contain new genes. The detection limit of DNA fragments on gels is approximately 10 nanograms (10 -9  grams). For a plasmid of 3000 base pairs (or 3kb), this means 3,000,000,000 copies (3 x 10 9 ), so if we have extracted potentially 10 11  – 10 12  copies of the plasmid, we have a sufficient amount to run a gel approximately 30-300 times and detect DNA bands each time.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px; line-height: 17px;">Plasmids can be genetically engineered because different plasmids contain unique DNA sequences called restriction sites. These are palindromic base sequences (usually 4, 6 or 8 base pairs in length) that are recognised by restriction enzymes, which endonucleolytically cut the plasmid, causing it to be linearised. Restriction enzymes are naturally synthesised in bacteria as protection from invading bacteriophage genomes. These enzymes are isolated from the bacteria and used commercially in restriction digests of plasmids for genetic engineering. The // E. coli // used for transformation are mutants in restriction enzyme synthesis: this is essential in ensuring that when they are transformed (see below) with the recombined plasmid, they do not digest the plasmid using their own restriction enzymes.
 * Engineering a plasmid **

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Digestion with certain types of restriction enzymes creates single-stranded sequences of unpaired bases called sticky ends. If the target gene and the plasmid vector are both digested with the same restriction enzymes, then these sticky ends will be complementary to each other and easily anneal. NCo1 and Xba1 are popular restriction enzymes in plasmid digests, because their restriction sites cover the start codon (ATG) and the stop codon (TAG) of genes, respectively. Restriction enzymes can also create blunt ends where there are no unpaired bases. Unlike sticky ends, these do not require sequence complementarity and blunt ends will anneal with any other DNA sequence. Although sticky and blunt ends can anneal themselves, ligation is secured by mixing the fragments and the plasmids in a test-tube with the enzyme DNA ligase and a supply of ATP. This forms covalent phosphodiester bonds between the sticky or blunt ends.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">It is important when digesting the fragments and plasmids not to //over-digest// them. Over-digestion can lead to the degradation of sticky ends because commercial stocks of restriction enzymes may also contain traces of exonuclease enzymes which have degradative ability. If these enzymes are allowed to degrade the sticky ends, then all the DNA will have blunt ends and because blunt ends ligate without sequence complementarity, it can be more difficult to get the target gene and the plasmid to ligate (as their sticky ends would otherwise have enabled).

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Once digested, all of the fragments should be run on an electrophoretic gel. The smaller fragment (target gene) should then be isolated for ligation with the larger fragment (the plasmid vector). However, there are a number of difficulties with this procedure. Firstly, electrophoresis does not have perfect resolution and so the gene fragment may be cross-contaminated with other fragments. When the gene fragment and the contaminating fragments are ligated into a plasmid, the resultant plasmid is called a // by-product //. Other, much more significant, sources of by-products are either plasmids that self-ligate (the sticky ends within the plasmid are also complementary and the probability of their ligation is higher than the probability of the target gene and plasmid coming into contact and ligating); or plasmids that were never digested in the first place (supercoiled plasmid DNA may bury restriction sites and thereby prevent digestion). So, three types of non-ideal 'by-product' plasmid are possible:


 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Those containing the gene fragment and contaminating fragments
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Those that have self-ligated with no foreign gene insert
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Those that never got digested in the first place

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">All of these plasmids can be incorporated into the E. coli and generate large colonies. This can make finding 'true transformants' as difficult as finding a needle in a haystack. The best way to ensure that the correct, recombinant plasmid has been assimilated is by isolating plasmids from colonies and checking them using restriction analysis. Another technique is the use of ligation controls.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12px; line-height: 17px;">Once a recombinant plasmid has been engineered, it needs to be re-introduced into a host organism for cloning. This is done by transformation. First the bacteria (usually // E. coli // ) needs to be made //competent// (i.e. capable of taking up exogenous DNA). This is done by growing the bacteria to the exponential phase, washing the colony with ice-cold calcium buffer several times, and then applying a heat shock of 37C for a few minutes. This causes the bacterial membrane to become permeable to DNA. <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">However this is a very inefficient process; approximately 1 in 100,000 plasmids are able to enter //E. coli// in this way. To increase the frequency of transformation events it is preferable to use more than 10 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%; vertical-align: super;">5 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> plasmids, around the order of 10 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%; vertical-align: super;">7 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">, and to have sufficient bacteria to act as potential transformants (usually 10 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%; vertical-align: super;">9 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> as a minimum). All of the bacteria are then plated out on a special medium for their subsequent growth and the replication of the plasmid. As mentioned above, the plasmid should be isolated from transformants so that it can be analysed by restriction digests, to ensure that it is in fact the recombinant plasmid that is desired.
 * Transforming a bacterium **

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Genetic markers are essential in determining which of the bacteria were successfully transformed. An empty E. coli strain (i.e. one that does not already contain plasmids) is used for transformation. The best genetic marker is to incorporate a gene for antibiotic resistance, such as resistance to ampicilline, into the plasmid along with the gene of interest. After the heat shock described above, the 10 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%; vertical-align: super;">9 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> cells are plated out evenly on a plate containing ampicilline. Most of the bacteria will die, but a few thousand colonies are formed after an overnight incubation. Such colonies are ampicilline resistant, because they each originated from a single bacterium that received a single plasmid with the gene for ampicilline resistance. These colonies are used to inoculate a liquid culture, and a small amount of the plasmid is extracted and purified (copies of the plasmid are retained because their extraction is difficult, and so it is useful to have a 'library' of them for future use).
 * Genetic markers **