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.

Recombinant DNA Technology

The aim of recombinant DNA technology involves cloning a specific gene. It is possible to produce multiple identical copies of a gene, a molecule, a cell through gene cloning. Recombinant DNA technology involves synergizing genetic material from different sources to create a new gene or a cell. Genetic engineering plays an important role in determining the structure of a gene, detecting carriers, diagnosing genetic disorders and gene therapy. Many hormones such as insulin, glucagon, and growth factors get synthesized using genetic engineering and used for commercial purposes. Before the advent of genetic engineering, there were limited sources for improvising crop quality, yield, agricultural techniques, and plant breeding.

However, with the recombinant DNA technology or genetic engineering, the yield and the crop quality started improving. Introduction of Stress and drought tolerance genes turned out to be successful. BT-Cotton is a classical example of recombinant DNA technology. It is a genetically modified crop involving resistance to bollworm infection. The plant gets modified with a specific gene helping the plant from bollworm attack. The gene product is a form of a protein. It gets released in the plant as soon as the worm attacks the plant. The worm immediately dies when it comes in contact with the gene product. Thus, BT-Cotton helped the farmers and the cotton industry grow. Many such success stories heard in the world involve the hard work of the recombinant DNA technologists. A body of techniques in recombinant DNA technology includes cutting the genes apart and splicing them with different pieces of DNA.

Image: Recombinant DNA technology

Materials used for recombinant DNA technology:

Recombinant DNA technology uses materials such as cells, bacteria, viruses, pieces of DNA, restriction digests, chromosomes, plasmid vectors, and many other substances. Commonly used organisms include bacteria as host organisms since they are single-celled structures. These bacteria are genetically engineered to produce vaccines, hormones, enzymes, and other biomolecules. For example, E. coli bacteria are genetically engineered to synthesize hormone insulin and growth factors. They also synthesize plant metabolites. An autonomously replicating DNA or a plasmid mostly depicts a circular, double-stranded DNA. Plasmids used in recombinant DNA technology replicate at each cell division. They are relatively convenient in isolation. Recombinant DNA technology allows the modification and manipulation of genes within or between the species. Hence, modification of plasmids involves restriction enzymes cleaving at a specific site and allowing foreign DNA inserts.

Recombinant DNA technology also utilizes viral vectors. However, it requires host genetic machinery for its replication.

Steps involved in recombinant DNA technology:

Cloning generates a series of DNA fragments. The first step involves cleaving the DNA segment at specific sites with the restriction enzymes. Restriction enzymes recognize and cut short sequences. They create a staggered or a blunt end in the DNA double helix. Now comes the role of a vector. A vector such as a virus or a plasmid carries a desired gene into the host and produces multiple copies.

Recombination:

Restriction enzymes cleave the DNA and produce either staggered or sticky ends. Thus, the plasmid DNA combines with foreign DNA. Their ends get sealed and stabilized through the action of another enzyme known as DNA ligase. The product obtained out of this activity is known as recombinant DNA. 

Transfer of the recombinant vector to the host:

The recombinant DNA molecules get introduced into the host organism. Once the recombinant plasmids get transformed into the host, they start multiplying themselves. The process develops identical foreign DNA molecules known as clones. A plasmid consists of genes showing resistance to antibiotics. The genes help the host to save themselves from the action of antibiotics. The absence of antibiotic resistance genes makes the bacteria sensitive to antibiotics. Screening of the clones involves nucleic acid hybridization.

References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu
[2] Biotechnology-4: Including Recombinant DNA Technology, S. Mahesh

© Copyright, 2018 All Rights Reserved.

Bacterial conjugation process

The review article focuses on the plasmid-mediated conjugation process in E. coli bacteria. A unidirectional transfer of the genetic material through a contact between the two bacterial cells is known as conjugation. A physical bridge between the two cells mediates the DNA transfer. In prokaryotes, such as bacteria, the transfer of the genetic material mostly involves a one-way process. Thus, the process of conjugation helps to transfer the genetic material from one cell to another, enabling the process of copying the genetic material. Among the bacteria, most widely used ones for genetic analysis involve Escherichia coli bacteria. 
Lederberg and Tatum first conducted a conjugation experiment on E. coli cells. William Hayes demonstrated the theory of the unidirectional transfer of the genetic material in E. coli. Bernard Davis independently conducted a U-tube experiment. With the help of the above studies, various researchers came up with different findings in bacterial genetics. Conjugation studies also help to map the genes. In the late 1950’s, Francis Jacob and Elie Wollman studied the transfer of genetic material from Hfr strains to F^- strains. Most of the conjugation occurs through plasmids in the bacterial cells.

A plasmid is an extrachromosomal genetic material present in the bacterial cell. These plasmids exhibit a property of transferring the genetic material. Hence they are used as vectors in the process of cloning and recombinant DNA technology. The contact between the two cells involves a physical bridge between the two cells. Thus, a segment of the chromosome from one cell transfers to another cell thereby undergoing genetic recombination. Hence, the cells receiving the DNA are known as trans-conjugants. The essential genetic element for a bacterial conjugation is known as a conjugon. Unlike prokaryotes, the process of conjugation in protozoa involves a two-way process.

Image 1: Conjugation in bacteria

Lederberg and Tatum experiment:

Two E. coli strains to differ in their nutritious environments were studied. Note that even bacteria require nutrients for carrying out various cellular activities.  The two bacterial strains were labeled as strain A and strain B respectively. The amino acid synthesizing bacteria do not require a supplemented medium. Such type of bacteria labeled as “+” strains, synthesize the required nutrients. The strain A had a genotype known as met bio thr⁺ leu⁺thi⁺. The strain A bacteria grew on a medium supplemented with methionine and biotin. Without these two functional molecules, the strain A would not have grown. The strain B had met⁺bio⁺thr leu thi genotype. It required threonine, leucine, and thiamine to grow. Both the strains were mixed and plated on a minimal medium. The mixed culture gave rise to the prototrophic colonies. No colonies were visible on the minimal medium after plating the strains individually. It is due to the auxotrophic cells.

A mutant organism capable of growing only on a minimal medium with the growth factor supplementation not required by the wild-type strains is known as an auxotroph. A strain of microorganisms not requiring any additional nutrient to grow is known as a prototroph. The prototrophic colonies occurred at a frequency of 1 in 10 million cells. These colonies were recombinants arising due to the exchange of the genetic material between the two cells.

Davis U-tube experiment:

Bernard Davis showed physical contact between the two bacterial cells using a U-tube apparatus. He placed both the bacterial strains in a liquid medium poured into either side of the tube separated by a filter. The medium moved between the compartments. It was later on, plated on a minimal medium. None of the colonies grew. Hence, through this experiment, Davis demonstrated the cell to cell contact of the bacteria mediated gene transfer.

William Hayes experiment:

The genetic exchange in the E. coli occurred in one direction. One cell acted as a donor and the other like a recipient. Sex factor or the F factor-mediated the transfer of the genetic material. There are two types of bacterial cells such as the donor and the recipient cells. The donor cells are the one giving the genetic material. The recipient cells accept the genetic material from the donor cells. F-factor is a plasmid capable of replicating independently. Hence, the donor bacteria are known as F+ strains. The recipient bacteria are known as F- strains. Two same types of bacteria do not undergo conjugation. The F+ and F- strains only undergo conjugation.

F+ and F- matings:

The process of conjugation involves mating between F+ and F- strains. The F factor of the donor bacteria has a nick at one of the strands extending through the sex pilli or a physical bridge. The nicked strand gets transferred to the recipient where the remaining strand gets copied. Hence, the transfer and the synthesis of the DNA gets completed. Once the transfer of the genetic material from F+ to F- strains gets completed, the F- strain with the genetic material now becomes a donor or an F+ strain. It becomes a donor with a very high frequency.

Three types of plasmids based on their mobility include conjugative, mobilizable, and non-mobilizable plasmids. A protein gets involved in the conjugative machinery. It is known as relaxase. It is an important protein capable of recognizing the origin of transfer (OriT). The OriT is a short DNA sequence required in the cis position. Relaxase catalyzes the initial and final stages of conjugation. It resembles rolling circle replication proteins. The mobilizable plasmids thus carry OriT, relaxase gene, and nicking auxiliary proteins. Though conjugative and mobilizable plasmids appear similar in their properties, still they exhibit a difference in the machinery required for gene transfer.

Hfr strain:

The high-frequency recombination strains (Hfr) originate by rare crossovers. The Hfr strain arises due to the integration of the F factor into the bacterial chromosome. Such type of F factors is known as episomes. Hence, it replicates as a part of the bacterial chromosome. The Hfr cells conjugate with the F- strains. The nicked strands in the integrated factor F get transferred to the recipient F- strain, thereby transferring the bacterial genes. The transferred strand gets copied along with the genes. Recombination occurs in the recipient. Though the genes get copied, an F- strain never acquires Hfr phenotype because a complete copy of the F factor of the Hfr strain does not retain. Only a part of the F factor gets transferred.

Occasionally, the Hfr cell may not be efficient in excision of the F factor. The host chromosome adjacent to the F factor sometimes gets integrated into it due to an aberrant excision. Not only one but many segments get aberrantly inserted into it. During this excision, the F factor plus bacterial genes loop out of the chromosome. It leads to the formation of F’ factor. This type of conjugation is known as F-duction or sexduction.  

Image 2: Hfr strain

Bacterial gene mapping using conjugation:

The interrupted mating experiment helped in mapping the bacterial genes. It involved a cross between the F – and Hfr strains.

1.     Hfr strain had genes such as Hfr H thr⁺ leu⁺ azi^R ton^R lac⁺ gal⁺str^s

2.     The recipient had genes such as F^- thr leu azi^S ton^S lac gal str^R

“S” indicates sensitive and the “R” indicates resistant. The generation of the recombinants results from a double crossover. At various time intervals, the conjugating pairs broke apart and the transconjugants plated on a selective agar medium. It helped in studying the gene transfer. A single F factor gets integrated into Hfr strain. The interrupted mating experiment revealed the circular structure of the E. coli linkage map.

What is an inter-kingdom conjugation?

Nitrogen-fixing bacteria undergo an inter-kingdom conjugation. Agrobacterium tumefaciens and Agrobacterium rhizobium undergo inter-kingdom conjugation. A few pieces of evidence also report the inter-kingdom conjugation between the bacteria and the yeast. Hence, it is not necessary for the bacteria to undergo conjugation between their species. Inter-kingdom gene transfer is an example of horizontal gene transfer between two species, or different organisms.

Applications in genetic engineering:

The transfer of the genetic material through the process of conjugation involves convenience. It is possible to transfer genes from one bacterium to another, from bacteria to the yeast, plants or other cells. With conjugation, it is possible to use or synthesize a metabolite. Conjugative bacteria show the ability to pick up new plasmids from the environment. Recombinant DNA technology uses plasmids as cloning vectors. 

References:
[1] Int Std Ed-General Biology, Peter Russel
[2] Genetics of Bacteria, Sheela Srivastava
[3] Introduction to Genetics: A Molecular Approach, Terry Brown

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