Shuttle vectors

Certain vectors act like a shuttle. A vehicle carrying people or goods regularly between the two places is known as a shuttle. Similarly, in the molecular world, certain vectors act like the shuttle. The shuttle vectors get transferred into two hosts. They move the desired genes between the organisms. Hence, shuttle vectors survive in more than one cell type. The shuttle vectors often shuttle between E. coli and yeast. Development of a shuttle vector involves plasmid isolation from the organism of interest and identifying its replicon. They have a very high transformation frequency. Recombinant plasmid libraries involve utilization of the shuttle vectors.

Components of a shuttle vector:

A shuttle vector contains separate origins of replication. Hence, these vectors have two origins of replication such as E. coli and yeast origins of replication respectively. There is a reason for having two origins of replication. The prokaryotic origins do not work in that of eukaryotic cells. Also, the required DNA sequence differs. Next, it should have a centromere sequence to allow the right partition of the plasmid in the yeast. It allows correct segregation of shuttle vectors at the time of cell division. The centromere sequence is known as a cen sequence. 
Multiple cloning site in the shuttle vector is nothing but a restriction site capable of getting cleaved with many restriction enzymes. Other genes present in a shuttle vector include antibiotic resistance gene and leucine biosynthesis gene. Selection of a plasmid for a yeast cell requires a leucine biosynthesis gene. The yeast cell starves without the adequate levels of leucine. Hence, the survival of the yeast requires the presence of leucine biosynthesis gene. Transformation of a shuttle vector into the yeast primarily involves rupturing the yeast cell wall using enzymes and calcium chloride. This step is important because the yeast cell wall consists of polysaccharide which creates a hindrance to the uptake of foreign DNA. Culturing the yeast in a suitable medium regenerates the cell wall.

Image: Shuttle vector

Introduction of a shuttle vector into the mammalian cells:

Designing of the shuttle vectors depends on the requirement of the mammalian cells. Later on, they get introduced into the mammalian cells and integrate with the mammalian chromosome. The mammalian shuttle vectors must have specific sequences associated with mammalian DNA. The first cloning experiment in mammals involved SV40 viral vectors. The SV40 viral vectors help the plasmid in replicating in the mammalian cells without integrating into the genome. The mammalian artificial chromosomes (MACs) consist of the telomeric and Centromeric sequences for segregation of markers.

Lambda shuttle vectors:

The transgenic animal experiments involve phage shuttle vectors. Hence, the animal cells readily accept the phage shuttle vectors. The transgenic animal undergoes a treatment with an exogenous agent leading to mutated reporter genes. Hence, the shuttle vector now contains the mutated genes. Detection of a mutated gene requires a single step in vitro packaging reaction. It involves a small amount of DNA prepared from a mouse tissue followed by extraction of shuttle vectors from the host. It gets packed into the infectious phage particle through in vitro packaging reaction. The bacteriophage shuttle vector gets integrated as a multi-copy concatemer. Treatment with a putative mutagen helps to remove the shuttle vectors from the chromosome with the in vitro packaging of the vector DNA into phage particle. The shuttle vector gets packed into the phage because of the presence of cos regulatory elements. Bacteriophage with shuttle vectors infect the bacteria followed by visualization of plaques. Mutated gene analysis involves blue-white screening of the colonies. An X-gal indicator compound distinguishes recombinants and non-recombinants.

Advantages of transgenic shuttle vectors:

They represent a cost-effective test for in vivo mutagenicity. It is easy to detect mutations in any tissue including the sperm and the ova. Shuttle vectors include commercial kits designed for various experiments.

Role of shuttle vectors in gene cloning by complementation of mutations:

A complementation test helps in determining the presence of mutants in a gene or a functional unit. The test distinguishes the wild-type and the mutant phenotypes. Two recessive mutations on the opposite chromosomes indicate a mutant phenotype. Hence, it results in no complementation. Two recessive mutations present in the same chromosome indicate a wild-type phenotype. Hence, complementation occurs. Two mutants present on the same segment arise due to positive complementation. A diploid or a heterokaryon contributes to studying complementation of genes. Gene cloning by complementation of mutations includes a classic example of yeast genes. It includes an example of yeast-E. coli shuttle vector. These vectors replicate autonomously and in the yeast and E. coli cells. The yeast-E. coli shuttle vectors consist of beta-lactamase gene, an ampicillin resistance gene, pUC 18 sequence, and multiple cloning sites. Loss of beta-galactosidase function indicates appropriate DNA insertion in the multiple cloning sites. A genomic library consists of DNA fragments obtained from wild-type yeast. A genomic library transforms a mutant host yeast strain. It consists of two mutations. One mutant helps in selecting the transformants. It is known as ura3. Another mutation helps in searching the wild-type gene mutation. The wild-type gene required for the biosynthesis of arginine is known as ARG1 gene. The mutation in the gene known as arg1 mutation helps in the complementation test. Arginine amino acid plays an important role in yeast cell growth. Yeast cells lacking arginine or enzyme responsible for arginine biosynthesis do not grow well.

Hence, arg1 mutant strains consist of an inactive enzyme for arginine biosynthesis. The shuttle vector helps in preparing the genomic library. It consists of URA3 as a selectable marker and yeast DNA consisting of the wild-type gene (ARG1 gene). The mutant yeast cells consist of ura3 arg1 mutant genes. Due to the transforming cells, some cells lacking the normal ARG 1 gene receive it from the genomic library having ARG 1 gene (which is wild-type). Such transformants express ARG 1 gene and synthesize arginine in their cells. Hence, they grow on a minimal medium in the absence of arginine in spite of having the defective arg1 gene. The ARG 1 gene overcomes the mutant arg1 gene by complementation. Hence, yeast-E. coli shuttle vectors helped in preparing genomic libraries for identifying complementation.

Yeast Integrative Plasmid (YIP 5) as a shuttle vector:

YIP 5 yeast vector consists of E. coli plasmid with a copy of URA3 gene. Hence, it is a shuttle vector used in both E. coli and yeast. It is difficult to clone the yeast cells and generate a large number of clones. Hence, the YIP 5 shuttle vectors solve the problem. The YIP 5 or a Yeast Integrative Plasmid consists of the ampicillin resistance gene (Amp^R gene), a tetracycline resistance gene (Tet^R gene), URA3 yeast gene and E. coli origin of replication. The size of the YIP 5 vector is 5.5 kb. Since there is an E. coli origin of replication, it is possible to construct recombinant YIP 5 molecules, before transferring them into the yeast cells. Without the origin of replication, the vector does not propagate independently inside the yeast cells but survives upon integration into a yeast chromosome. Homologous recombination between the URA3 gene in the vector and the chromosomal copy of the same gene results into the integration. Hence, it replicates along with the host chromosome.

Yeast episomal plasmid vector:

A high copy number plasmid gets combined with another plasmid part known as pBR322 for constructing a shuttle vector. A yeats episomal vector consists of segments from pBR322 carrying origin of replication, ampicillin resistance gene, a selectable marker gene of yeast, and REP gene for replication.

Animal viral vector:

The growth of the basic animal vector involves an E. coli cell because of its convenience. The mammalian vectors are shuttle vectors having prokaryotic sequences for the propagation of the vector in E. coli and expression units of eukaryotes (mammals). 

References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Genetics, 9th Edition (Multicolour Edition), Verma P.S. & Agarwal V.K.

[3] Gene Cloning and Manipulation, Christopher Howe

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] IGenetics, Peter Russell

© Copyright, 2018 All Rights Reserved.

Cloning vectors

Image 1: Cloning vectors

The concept of cloning vectors is similar to the example described below. Imagine you want to reach a destination far away from your house. So, you use a vehicle to reach that place, isn’t it? The vehicle carries you to the desired destination. In the molecular world, the extrachromosomal DNA known as the plasmids act as vehicles in carrying the fragment of interest to the desired location. Thus, in DNA cloning, a plasmid or phage chromosome acts as a vector (vehicle) to carry the cloned DNA segment. There are many types of vectors such as plasmids, cosmids, phagemids, shuttle vectors, expression vectors, and artificial chromosomes.

1.   Plasmid vectors:

The extrachromosomal elements present in the bacterial cells replicate autonomously or independently. Plasmids have a circular DNA with double strands. It consists of sequences capable of independent function. Naturally prevalent plasmids serve as the main source of plasmid vectors. They are engineered to get the desired features. Plasmid extraction is easily possible with the E. coli bacterial cells. These plasmids pass from one cell to another cell. Replication of plasmids occurs during each cell division. We obtain plasmids through disruption of the bacterial cells followed by centrifugation. Next step involves treatment with the restriction enzymes. Cleavage of a plasmid produces staggered ends. The foreign DNA gets incorporated in the vacant site. Note the two important points about the plasmids. Firstly, the plasmids possess a limited number of restriction sites. Secondly, the plasmids carry genes for resistance to antibiotics. Care must be taken to avoid incorporation of any harmful gene into it. 

Image 2: Plasmid Vector

The features of E. coli plasmids are as follows:

·        Origin of replication: The site for starting or initiating the replication is known as the origin of replication. It permits an independent replication of the plasmid.

·        Selectable marker: It is usually dominant. A selectable marker gets readily detected. Thus, it exhibits a trait suitable for artificial selection. One more type of marker is known as a screenable marker. It allows the researchers to distinguish between the required cells and the unwanted cells. An antibiotic resistance gene acts as a selectable marker. Examples include ampicillin or a tetracycline resistance gene. The positive and negative markers are the two types of markers. The markers conferring a selective advantage to the host are known as positive markers. The markers eliminating the growth of the markers are known as negative markers.

·        Restriction enzyme cleavage sites: The restriction sites are present just once in a vector. The DNA fragments get inserted into the vector at specific sites. An appropriate restriction enzyme recognizing this sequence gets selected for cleaving that particular site. Later on, the fragment of interest gets inserted into that site. Some of the plasmids have multiple cloning sites. Plasmids act as disease models. Protein productions and gene therapy also involve the use of plasmid vectors.

Example of a plasmid vector: pUC 19 vector

pUC 19 or “puck” 19 vector is a 2686 base pair vector with multiple cloning sites. They are unique restriction sites clustered in one region. A high copy number of the plasmids and amp^R selectable marker make it a good choice for cloning experiments. The polylinker has restriction sites for various restriction enzymes such as Eco RI, Bam HI, HindIII, PstI, SacI, Sma I and many other enzymes. The polylinker is within the beta-galactosidase gene (lac z+). The pUC 19 vector gets cleaved at its polylinker site leaving a vacant space. Next, a piece of DNA to be cloned gets separated through electrophoresis and obtained for further procedures. The piece of DNA, the vector, and the ligating enzymes are mixed. The DNA gets inserted into the plasmid and the gaps sealed by DNA ligase. The resulting plasmid gets transformed into E. coli. It involves incubation of the recombinant plasmids with E. coli cells and treating chemically or by electroporation. Next step includes plating of the transformed cells on a suitable medium to observe the colonies. Blue-white screening distinguishes recombinant DNA clones from the plasmids. Episomes are the plasmids capable of integrating into the chromosome.

2.   Phagemids or Phasmid:

It is a cloning vector with two properties such as bacteriophage and plasmid properties. They are DNA based vectors. These phasmids exhibit a property of getting packed into phage heads.

3.   Fosmids:

They resemble cosmids and involve bacterial F plasmids. E. coli serves as a source of fosmid vectors and contains only one fosmid.

4.   Shuttle vectors:

The vectors introduced into two or more organisms are known as shuttle vectors. For example, some vectors get transformed into E. coli and also get transformed into the yeast. These plasmids propagate in the prokaryotes and eukaryotes. Example, an Adenovirus shuttle vector propagates into both prokaryotes and mammals. Multiple copies of a gene get synthesized. A shuttle vector consists of two origins of replication specific to each host. Shuttle vectors are also known as bifunctional vectors. A yeast shuttle vector consists of bacterial origin of replication, yeast origin of replication, yeast selectable marker, bacterial selectable markers, and an origin of transfer.

5.   Cosmids:

Cosmids are also known as hybrid plasmids. They consist of a lambda phage cos sequence. The genetic engineering technique uses cosmids as cloning vectors. They have a suitable origin of replication and an antibiotic resistance gene. These plasmids get packaged into phage capsids thereby allowing transduction. The cos sequences present in a cosmid are just a few hundred base pairs in length. The availability of the cosmids as the shuttle or mammalian cosmids depends on the need of the experiment. A typical feature of the cosmid involves the formation of colonies instead of forming plaques.

6.   Expression vectors:

A plasmid or a virus behaves like an expression vector. However, an expression vector designing must boost the expression of the desired gene. The gene of interest gets inserted into the expression vector. The gene contains a protein-coding region. The expression of the desired gene produces a protein product.

Image 3: The Expression vector

7.   Plant vectors:

The plant vectors involve Ti-plasmid vectors. The Ti-plasmids belong to a soil bacterium known as Agrobacterium tumefaciens. It consists of T-DNA capable of integrating into a plant genome. Integration of plasmid into the plant genome occurs when the bacterium infects the plant and causes crown gall disease. An example of plant cloning vector involves the pBIN vector. It consists of a lac z’ gene, a right T-DNA boundary, a left T-DNA boundary, an origin of replication, and kanamycin resistance gene.

8.   BAC’s and YAC’s:

The bacteria exchange their plasmids through a process known as conjugation. The donor bacterial cell transfers its F plasmid through a cellular bridge. BACs or bacterial artificial chromosome construction involves F-plasmids. The human genome project utilized bacterial artificial chromosomes for various projects.

·        Components of BACs:

·        oriS, rep E-F: It is the origin of replication. It regulates the copy number of the plasmids.

·        The parA and par B components help in the partition of the cells into daughter cells during cell division.

·        Antibiotic resistance gene acts as a selectable marker.

·        Phage promoters induce transcription of an inserted gene such as T1 and Sp6 promoters.

Various disease studies involve BACs as models. A BAC library is a collection of BAC clones stored in regulated temperatures. Several genes and large gene studies involve BACs. They are useful in the development of vaccines.

Yeast artificial chromosomes or YACs help in cloning large DNA thereby guiding in an accurate construction of a physical map. They involve genetically engineered yeast DNA.

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2]Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh

[3] Gene Cloning and Manipulation, Christopher Howe
[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

© Copyright, 2018 All Rights Reserved.