Genetic Engineering is the technique of biotechnology which helps in preparing recombinant DNA. DNA molecule is cut into small pieces in in vitro environment. There are numerous techniques which have been used in genetic engineering for example, recombinant DNA technology, microinjection, bioballistics, electro and chemical poration.
Following steps are involved in recombinant DNA technique;
1) Gene of interest is isolated from the DNA molecule using the restriction enzymes.
2) After isolation, gene is inserted into a vector and is cloned to make multiple copies of gene of interest.
3) When the cloning is done, the gene is incorporated into the plasmid.
4) Now the gene or DNA along with the plasmid is called as recombinant DNA.
Plasmids and vectors used for the recombinant technique are usually bacteria or viruses. They have the ability to carry foreign genes into the host cell where they release the gene of interest and this gene replaces the diseased gene.
Plasmid is a circular piece containing the genetic material. When new gene is inserted in it, it opens it ring and new gene is attached to its ends through the enzyme called as ligase. New gene replicates along with the plasmid’s genetic material. For example if plasmid is carrying a gene of insulin, t will start producing the protein of insulin along with other gene products. Bacteria are of great significance in the pharmaceutical industry because they are used to produce insulin and other useful proteins.
Vectors are usually viruses which are also helpful in genetic engineering. Virus is an infectious organism, so when a new gene is inserted in it, it transfers that gene into the host cell during causing infection. Scientists mostly block the function of virus when they insert the foreign gene; this way virus will only be able to replicate only the gene of interest and will insert it into the host cell.
Electro and chemical poration:-
In this method, pores are created in the membrane of the cell and genes can be transferred easily. Special chemicals are used to make pores in the cell surface. Sometimes cells are exposed to weak electric current, it also makes pores in the surface of the cells and genes can easily pass through these pores.
In this method, small silver particles are used to insert the genetic material into the recipient cell. These silvers are coated with the genetic material and when released in the cell, genetic material incorporates with the genes of the host cell. In one projectile method, shot gun is used to insert the silvers into the host cell.
It is not necessary that only plasmids and vectors should be used for the transfer of genes into the cells. There are methods which are not dependent on plasmids and vectors. One of these methods is microinjection. In this method, foreign gene is integrated into the cell by just injecting it into the recipient cell. When large cell of plants and animals are concerned, then a fine glass needle is used. The injected genes automatically enter into the nucleus where they incorporate with the host cell’s genetic material and replicate.
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Vector is the carrier molecule which is used in genetic engineering in order to transfer our gene of interest into the host cell where it will be expressed. There are a lot of reasons for this action ranging from the study of gene expression to the production of needed proteins. Once the vector has accepted our DNA of interest, it becomes a recombinant molecule.
There are several characteristics which are important for the vector molecule in order for it to be efficient and worthy of using in genetic engineering.
It is very important that vectors are small molecules (relatively speaking), because this way it will be a lot easier to insert them into the host cell, making the whole process of transformation (insertion of vector molecule into the competent host cell) a lot more efficient.
It has to have an origin of replication. This is the region on the DNA that will be recognized by the protein complex responsible for the initiation of transcription. Without the origin of replication, our gene of interest will not be expressed in the host cell.
The process of transformation in not very efficient (depending on the approach), so if we are working with the certain amount of bacterial cells that we want to transform, not all of them will accept our vector (and gene of interest). In fact, probably most of them won’t. How can we know which cells have our vector inside? It is simple - by using vectors with selectable markers which will help us distinguish the cells that have successfully transformed from the ones that haven’t. For example, we can use a certain vector molecule which has the gene for ampicillin resistance in it. Then we can grow the cells in the cell culturing medium which contains the ampicillin. Only the cells that are transformed successfully will be able to grow in the medium, because we will eliminate all of the other ones in a simple and effective way.
How can we insert our gene of interest into the vector molecule? We can accomplish this by using restriction enzymes which cut the DNA at specific places. These places are called target sites and they are usually short, palindromic sequences compatible only with specific restriction enzymes. Once they cut the DNA, our gene of interest can be inserted and sealed in the vector molecule using the enzyme ligase, for example.
This is the division relevant to the genetic engineering and transforming target cells. There is also another division of plasmids according to their function (like resistance plasmids, degradative plasmids, virulence plasmids, etc.), but this is not really important for the subject, so I won’t cover it here.
Low-copy number plasmids
The main feature of these plasmids is that they replicate in low numbers in host cell. This enables the metabolism of a host cell to stay at low level, enabling the cell to reproduce moderately. They are easily constructible and they can be used to study genes in basic metabolic pathways, or to clone genes whose overexpression can kill the cell. They are also known as stringent plasmids, as their replication is “bounded” to, or limited by the replication of the host cell DNA.
Subtype of low-copy plasmids are runaway plasmids. They are sensitive to temperature and their expression (replication) remains low in the optimum temperature. If the temperature increases too much, they get overexpressed, which results in the death of the cell.
High-copy number plasmids
Opposite from low-copy number plasmids are high-copy number plasmids. The name tells for itself – they are able to replicate in high numbers after they enter into the host cell. They are also known as relaxed plasmids, because they are not limited by the replication of the host cell DNA. They can be used for the production of a certain protein which is needed in high ammount.
These plasmids contain mob (mobilizing) and tra (transfer) genes which help them transfer to the bacteria on their own. Tra genes are responsible for the formation of F-pilus – a bridge between two cells through which the plasmid can transfer itself. They usually have high molecular weight and they replicate only into few copies (1 to 3 per cell). Example of conjugative plasmids is F-plasmid.
These plasmids are the opposite of conjugative plasmids. They do not contain mob or tra genes and are therefore incapable of initiating the process of conjugation, so they need help from conjugative plasmids in order to be transferred. They have low molecular weight and they usually exist in multiple copies per cell.
These plasmids are somewhere in between of conjugative and non-conjugative ones, because they contain mob genes, so they can use conjugative plasmids in order to transfer to other cells.
06-17-2014, 02:08 AM
(This post was last modified: 06-18-2014, 02:50 AM by zemaxe7.)
Bacteriophages are basically bacterial viruses (eaters of bacteria), and they usually consist out of DNA genome enclosed in a protein head, also known as capsid, made out of protein icosahedral (except in filamentous phages; their genetic material is not encapsulated by proteins). They do not exist as free-living organisms and they depend on bacteria for their propagation. Vectors based on the viruses are called bacteriophage vectors. There are three types of them based on the structure:
- Bacteriophages with both head and tail
- Bacteriophages with only head
- Filamentous bacteriophages
Their genetic material can be either double stranded or single stranded DNA or RNA, even though cases where genetic material is based on RNA are rare. Their genomes usually represent 50% of the whole weight. Adsorption is the term used for the attachment of the virus to the surface of the cell which results in the viral DNA being injected into the cell.
Bacteriophages can also be divided based on the lifecycle:
- Virulent – exhibit lytic cycle
- Temperament – exhibit lysogenic cycle, even though they can sometimes shift to lytic cycle
During the lytic cycle, there is no integration of viral genome into the bacterial genome. Instead, the viral genome replicates independently, host cell’s genome degrades and the cell eventually dies producing additional phages.
During the lysogenic cycle, after the adsorption, the virus inserts its DNA into the cell and integrates itself into the host cell’s genome, becoming a prophage (inactive virus, integrated into another genome). This allows for the normal replication of the host cell to happen.
Cycle that is going to occur depends on the nutritional and bacterial metabolism state as well as on the MOI (multiplicity of infection), which is basically the ratio of viruses to bacteria during the infection. Cycle change from lysogenic to lytic can also occur under certain conditions (if the cell is exposed to UV light, for example).
Vectors Based on Bacteriophage Lambda
Lambda phage can be used in genetic experiments as a vector because not all of its genome is essential for its function. This allows for the introduction of new exogenous DNA if certain requirements are met:
- Arrangement of the genes on the phage’s genome determines which parts can be removed in order to add our DNA of interest. Luckily, no complex in vitro rearrangement is needed because the central region of the lambda genome is dispensable since it controls the lysogenic properties and we need lytic one in order to successfully transfect the target cell.
- Restriction sites have to be taken into consideration since the wild type lambda phage has a lot of them which represents a problem because it limits the choice of sites for the insertion of the DNA. This can be solved by simply choosing the phage which has reduced number of sites for particular restriction enzymes. The rest of the restriction sites can be dealt with using the technique of mutagenesis in vitro, which will modify them and make them unrecognizable by restriction enzymes.
Lambda phage can be used as insertion vector, without cutting any part of its DNA out. However, this will result in the smaller DNA insert, especially since the capsid limits the size of the DNA which can be inserted to only about 2.5 kb. Insertion vectors have only one restriction site which enables DNA fragments to be inserted into their genome. λgt10 and Charon 16A are some examples of lambda-based insertion vectors, and they can accept a bit more exogenous DNA, 7.6 kb and 9 kb, respectively.
In order to increase the amount of DNA which can be inserted, scientists have developed replacement vectors which have a stuffer fragment inside themselves that can be removed, thus providing more space for our DNA of interest. Replacement vectors need at least 2 or more restriction sites, and some examples of them are EBML4 and Charon 40.
Vectors Based on Bacteriophage M13
Phage M13 has some advantages but also some disadvantages concerning the usage for genetic experiments and based on them, it should be chosen only in some cases.
The first good thing about M13 is that its RF (replicative form) is similar to plasmid, so it can be isolated and manipulated using the same techniques. Moreover, single-stranded DNA of M13, which is produced during the infection, is useful in techniques like DNA sequencing by dideoxy method. Sequencing is very important in genetic experiments, and this feature of M13 bacteriophage makes it a good target for a potential vector.
The difference between phage lambda and M13 is in their genome. M13 does not have any non-essential genes making this one of its greatest downsides. Moreover, its genome is filled up very effectively, so its intergenic region (available for manipulation) is only 507 base pairs long. However, its genome is already pretty small (6407 base pairs).
Intergenic region in M13 is used to insert a polylinker/lacZ sequence into the vectors based on M13, which enables the X-gal screening system (blue white selection) making detection of recombinants easier. When M13 is grown on a bacterial lawn, plaques appear because of the reduction in growth of host cells, which can be picked up for further analysis.
Another disadvantage of vectors based on bacteriophage M13 is the amount of exogenous genetic material they can accept. Even though the capsid of M13 is actually determined by the size of its genome, it still cannot accept large fragments of exogenous DNA. In fact, anything longer than 1.5 kb is considered to be too much for M13 based vectors because it makes them loose their cloning efficiency. This problem is bypassed simply by using M13 vectors for cloning and sequencing of small DNA fragments; especially in the situations where the ease of purification is needed, or when potential sequencing is planned later on.
Plasmids and phages are “regular” types of vectors that have not been heavily modified. However, the advancements in our technology and the need for more complex genetic experiments have led us to create new, improved vectors. This is due to several reasons. First of all, genetic experiments started shifting from cloning of single or several genes to the cloning of whole genomes. Secondly, cloning procedures have been commercialized to make them more available for “regular folks” through cloning kits. Nowadays, you don’t have to be awesome scientist in order to perform some decent genetic experiment (if you have the money, that is).
Some of the new vectors developed for engineering purposes are also hybrid vectors. These vectors, like cosmids or phagemids, incorporate features from different vector types (from plasmids and phages, in the case of cosmids and phagemids).
These are vectors made of plasmid sequences joined up by phage cos sites. Cos site represents several bases at both ends of the linear phage genome that are complementary and are able to circularize its DNA once it is inserted into the host cell (they are essentially sticky ends). The base of cosmid vectors is really small (around 4-6 kb) allowing them to accept relatively large amount of exogenous DNA (up to around 45 kb). Since they do not really have phage genes, they behave as plasmids, except that their insertion mechanism is the one from lambda phage. Basically, they are very efficient and are able to take a lot of foreign DNA, but they need more complicated procedures and their cloned sequences need further processing.
These hybrid vectors are based on the phages, giving them some advantages over cosmids by utilizing phage functions. Basically, they have f1 origin of replication “borrowed” from the f1 filamentous phage (it is in the same group as the phage M13). They are somewhat better because they have the ability to excise cloned DNA fragments in vivo as part of a plasmid, which removes the need for further processing of cloned sequences.
Just when I thought I had forgotten the techniques I manage to read this post and it refreshed my memory completely! I wonder how many more methods are going to come up within the next few years.. if they can introduce genetically modified material in any other ways. It would be interesting to see!
This is interesting enough for now though, lovely set of posts! Very informative!