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Techniques of Genetic Engineering
#5
Bacteriophage Vectors

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).
#6
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.
#7
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.
#8
Hybrid Vectors

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).

Cosmids

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.

Phagemids

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.
  

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