Vectors

VECTORS:

The carrier DNA molecule into which the desired DNA fragment is integrated is called as vector. A vector should have three essential features

1. It should be able to replicate in host organism by itself and also along with the insert DNA.
2. Its presence or absence in cell or organism should be easily identified.
3. In some vectors where the interest is in production of RNA or protein, it should be able to express the insert DNA.

Origins of replication: These are the sites where replication initiation occurs. Origins are AT rich sequences that undergo unwinding easily to facilitate the initiation of replication. (Promoters are also AT rich sequences where initiation of transcription occurs). Any DNA molecule containing an origin will be capable of undergoing replication by itself in a specific host.

A DNA region containing an origin and a terminator is called a replicon. Replicons are units of replication.Prokaryotic genomes eg: E.coli contains a single origin. Therefore they are called as single replicons. In case of eukaryotes each chromosome contains 1000-10000 origins, Eukaryotes contain multiple replicons so as to reduce gestation period..

Origins are host specific. Origins vary from one host to another, for instance, in E.coli the origin is col E I. A vector containing col E I origin may not replicate in Bacillus. In yeast ARS (Autonomous replicating sequences) act as origins. Origins regulate the copy number of a plasmid. Copy number is the number of vector molecules present in the cell or organism at a given time. Since origins control the replication initiation process they also decide the copy number. Vectors which replicate many times and exist in large numbers (1000 molecules per cell) are called as High copy number vectors. Eg: plasmid derived vectors.

Vectors which replicate only once or twice and exist in low no. (1-5 molecules) per cell are called as Low copy number vectors. Eg: BAC vectors and YAC vectors.

Plasmid incompatibility: Origins also control the compatibility of vectors in a cell. Two vectors i.e. plasmids containing similar origins cannot co exist in a cell.

Selection markers:

Selection markers enable the differentiation between transformants and non transformants. Transformant is a cell or organism which has received external DNA by transformation. Second or additional section marker enables the differentiation between recombinants and non recombinants. Selection markers are often the genes which confer the antibiotic resistance.

Eg: bla gene – it encodes for ß-lactamase which confers resistance against the antibiotic ampicillin. Ampicillin inhibits cell wall synthesis. It is a bacteriostatic antibiotic. Ampicillin ( similar to pencillin) contains ß-lactome ring. An enzyme called as ß lactamase produced by the bla gene cleaves the ß lactum ring of ampicillin and inactivates it. Therefore cells which have received the DNA (transformants) can survive and grow in media containing ampicillin as selection agent. On the other hand cells which does not receive the DNA (non transformants) fail to grow and develop into colonies in media containing ampicillin.

Appearance of star colonies: Media plates containing ampicillin plated with transformation mixture (transformants + non transformants) develop/show colonies upon incubating for 12-16 hrs at 370C. Sometimes upon over incubation (more than 16 hrs) small sized colonies appear around the region of large transformed colonies and are called ‘star colonies’. These small colonies appear in the area around large colonies where ampicillin has been inactivated by the production of ß lactamase produced by transformed colonies. They appear only after the degradation of ampicillin and hence take more time.

Replica plating:
In order to differentiate between recombinants and non recombinants, a second selection marker is required. First selection marker will be intact in both recombinants and non recombinants. Second selection marker contains restriction enzyme sites where the insert DNA is integrated. Due to the insertion of insert DNA, the reading frame of the selection marker gene gets altered and hence gets inactivated. This phenomenon is called as Insertional inactivation.



Due to this, the second selection marker is non functional in recombinants and functional in non recombinants. Upon transformation, transformants appears on the plate containing first selection agent, for example - ampicillin. These colonies are imprinted onto another plate containing second selection agent, for example - Tetracycline, by using a filter paper or a sterile cloth. This process of imprinting the colonies from one plate to another is called as Replica plating. Upon replica plating only some of the colonies which earlier developed on ampicillin containing plates grow on tetracycline plate. These colonies which develop on tetracycline containing plate are the non recombinants; Non recombinants grow both on tetracycline and ampicillin plates. Recombinants grow only on ampicillin plates but not on tetracycline plates.

Types of selection markers:
There are two types of selection markers

1. Screenable markers
2. Scorable markers.

Screenable markers:
Antibiotic resistance conferring genes fall into this category. These markers require a screening procedure like replica plating to differentiate between recombinants and non recombinants.

Scorable marker:
These markers does not require any additional screening procedure and based on the phenotype (colour) transformants can be differentiated as either recombinants or non recombinants.

Eg: Lac Z a fragment

a Complementation :
Complementation occurs for a region having different mutations in different loci. Restoration of a lost phenotype due to mutation is called as Complementation
The E.coli strains that are used in molecular biology contain many mutations. These mutations do not allow the organism to survive independently in natural environment. This is called as Biosafety. In E.coli, the lac operon contains a deletion mutation in the a fragment coding region of lac Z gene which codes for ß- galactosidase. ß- galactosidase is a tetramer consisting of two a and two ß subunits. As the mutation in a region these E coli strains cannot produce functional ß galactosidase enzyme. When such strains are transformed with vectors containing lac Z a portion as second selection agent, it leads to restoration of lost phenotype. The lac Z a portion from the vector codes for functional a subunit, which combines with functional ß subunit coded by the lac Z gene present on the chromosomal DNA of E.coli. This results in the formation of functional ß galactosidase enzyme and since the mutation present in lac Z a portion of the E.coli chromosomal DNA is complemented by wild type lac Z a region on the vector DNA , it is called a complementation.



Blue-White selection:
X – gal is a lactose analogue which is 5-bromo 4-chromo 3-indolyl ß galacto pyranoside. X- gal is a chromogenic substrate i.e. it produces blue color upon cleavage by ß galactosidase. Cells transformed with vector DNA exhibit a complementation and hence produce blue colored colonies. On the other hand, cells transformed with recombinant vector cannot produce functional ß galactosidase enzyme due to insertional inactivation and hence produce colorless or white colonies. Therefore upon transformation recombinants and non recombinants can be differentiated based on the color of the colonies. Blue colored colonies are non recombinants and white colored colonies are recombinants. This is called Blue-White selection.

Positive selection markers:
The vectors which contain toxic genes as selection markers exhibit Positive selection. Cells which receive non recombinant vector will be killed due to the production of toxic protein. Cells which receive recombinant vector can only survive because the toxic gene will be insertionally inactivated.

Eg: ccd genes and sacB gene in E.coli and pmi gene in RIce

Promoters and terminators:
A promoter is a region where RNA polymers can bind and accomplish transcription. RNA polymerase can bind to any region of the DNA by means of general affinity, this is called as loose binding. When RNA polymerase core enzyme a2ßß’? combines with sigma factor, its affinity for promoter region increases and therefore specifically binds to promoter region. This is called as tight binding. Promoters are AT rich sequences which undergo easy denaturation to provide single stranded DNA template to promote transcription The region of DNA where RNA polymerase dissociates from the template DNA releasing the nascent RNA molecule is called as terminator.

Ideal features of a vector:

1. Small size – It is preferable that vectors are relatively small in size which enables easy handling
2. Selection marker- Having more than one selection marker enables easy differentiation of recombinants and non- recombinants
3. Multiple cloning site: It is the region of the vector which contains a continuous sequence of unique restriction enzyme recognition sites

Types of Vectors:

1. Cloning vector: It is a vector which can replicate by itself and also along with insert DNA. Such vectors contain origins and selection markers. Purpose of cloning vector is to produce number of copies of insert DNA
2. Expression vectors: Vectors which contain promoters and terminators in addition to origin and selection markers are called as expression vectors. The purpose of expression vectors is to produce RNA or proteins of interest
   
   
3. Promoter probe vectors: These vectors are used for testing the functionality of DNA sequences to be able to act as promoters. Such vectors contain origin, selection markers and reporter genes followed by terminator but lacks promoters. DNA sequences to be tested as promoters are ligated upstream to the reporter gene. In case of the ligated DNA functions as a promoter, transcription and translation takes place leading to the formation of reporter proteins

Reporter genes:
It reports the expression levels of the protein. They are used to test the function and strength of the promoter

1. Lac Z codes for ß galactosidase which upon acting on X-gal produces blue colour. The amount of enzyme produced is directly proportional to the no. of substrate molecules cleaved which can be estimated by the intensity of the blue colour formed colorimetrically
2. GUS codes for ß glucuronidase which acts on glucutonic acid analogue X-Gluc and produce blue colour
3. CAT (Chloromphenicol acetyl transferase) the no. of acetyl chloromphenicol molecules formed can be measured by chromatography
4. GFP (Green fluorescent protein) this gene is isolated from jelly fish Aquarea Victoria. This protein has auto fluorescence ability. This protein can be used for identifying intracellular localization of desired protein when GFP is joined to it.
5. Luciferase This gene has been isolated from firefly. It acts on luciferin and emits light

Six different types of cloning vectors:

1. Plasmid cloning vectors
2. Phage l cloning vectors
3. Cosmid cloning vectors
4. Shuttle vectors
5. Yeast artificial chromosomes (YACs)
6. Bacterial artificial chromosomes (BACs)

Plasmid cloning vectors:
Plasmids are extrachromosomal, autonomously replicating DNA molecule which are usually double stranded and are dispensable molecules. Plasmids contain their own origin of replication

Origins regulate the copy number of plasmids. High copy number plasmids contain origins which exhibit relaxed type of control for DNA replication. Proteins required for replication if the plasmid are mostly encoded by the plasmid itself such plasmids undergo replication several times independent of chromosomal DNA replication. Plasmids with low copy number.exhibit stringent type of control for DNA replication. These plasmids require some proteins encoded by chromosomal DNA for their replication. Therefore plasmid replication is dependent on chromosomal DNA replication; hence they replicate only once or twice and are present in low numbers.

Inability of two plasmids to coexist together in a single cell is called Plasmid incompatibility. Plasmid incompatibility is a consequence of having similar origins.

Par regions: are responsible for equal distribution of plasmids into daughter cells during cell division. Lack of par regions leads to segregation instability (improper/ unequal distribution of plasmids.

Linear plasmids:
Plasmids are usually double stranded and circular in shape but linear plasmids have been discovered in Borrelia burgdorferi and Streptomyces lividans.

Plasmid curing:
The process of removal of plasmids from cells is called as plasmid curing. Plasmids are not absolutely essential for the survival of the cell. Presence of plasmids gives selective advantage to the cells under specific conditions.

Antibiosis: It is a phenomenon where bacteria produceS toxic substances to kill other bacterial species. These toxic substances are called as antibiotics. Genes which code for antibiotics are present on plasmids.

Resistance against antibiotics is widely spread by horizontal gene transfer because of plasmids and transposons. Plasmids contain gene for mediating conjugation. Tra genes are responsible for transfer of DNA from F+ cell to F- cell.

Plasmid also contain genes for providing resistance against heavy metal toxicity toluene utilization.

Plasmid derived vectors:
Although natural plasmids can act as vectors they lack unique restriction enzyme sites and are usually large in size. Therefore plasmids are engineered to possess features of an ideal vector. Such vectors are called as plasmid derived vectors The first plasmid derived vector developed was pBR322 (B-Boliver R-Rodriguez). pBR322 is a low copy no. plasmid which is large in size and contains less no. of unique restriction enzyme sites.



Next plasmid derived vectors are developed at university of California called pUC 8 and 9. These vectors are relatively small in size, contain a high copy number, origin and more unique sites. pUC 8and 9 differ in the orientation of MCS (opposite direction).Plasmid vectors become unstable when their size is more than 10 kb.

Phagemid vectors:
Plasmid vectors containing DNA elements of bacteriophages especially origins are called as phagemid vectors. M13, f1 and f9 DNA containing vectors. Origins from M13 or f1 or f9 are incorporated into plasmid vectors upstream of MCS (5’end)..DNA replication from these origins leads to the production of single stranded DNA which is used for DNA sequencing and generation of probes.

Cosmid vectors:
Plasmids containing lambda cos sites are called Cosmids. Cos sites are sticky or cohesive ends which are easy to ligate.. These vectors contain the features of both plasmid vectors and lambda vectors. Features of both plasmid and phage cloning vectors. They do not occur naturally and are circular in shape. They possess Origin (ori) sequence for E. coli, selectable marker, e.g. ampR and restriction sites. Phage l cos site permits packaging into l phages and introduction to E. coli cells. Its insert capacity is 35-45kb.



Lambda vectors:
Lambda is a Temperate phage with double stranded DNA genome. The genome is a linear DNA molecule of length 45.5kb. At the end of linear molecular single stranded overhang regions of 12bp are present which are complimentary to each other. These overhang regions are known as cos sites. The linear DNA molecule after getting introduced into bacterial host gets circularized because of cos sites as circular DNA cannot be cleaved by exonucleases.



From the lambda genome of 48.5 kb some nonessential regions are eliminated to reduce its size. The lesser sized DNA molecule is engineered into a vector to combine with insert DNA. The capacity of lambda vector ranges from 5-25kb. Lambda vectors like other viral vectors have the following additional beneficial features like In vitro packaging. The recombinant DNA (Lambda vector + insert DNA) when combined with core proteins can form a viral particle spontaneously. Therefore any insert DNA can be combined with lambda vector and can be packaged as lambda phage. Viral vectors have longer shelf life than plasmid vectors have longer shelf life and can be stored as such whereas bacterial cultures are mixed with glycerol and stored at -80°C for longer duration. Glycerol acts as cryopreservative agent (anti freezing agent). In case of animal cells DMSO is used as cryopreservative agent.


BAC vectors:
Bacterial artificial chromosomes are designed based on features of F plasmid. The natural function of F ’ (F prime) plasmid is to allow a bacterium to transfer its chromosome to another bacterium. It contains origins for replication and par regions. Their insert capacity is upto 300kb. These vectors exhibit stringent type of replication control and exist as low copy number vectors being found in one or two copies per cell reducing the risk of recombination between fragments.

Advantages of BAC vectors:

1. BAC vectors can be transferred to E.coli by electroporation
2. Transformation efficiency is high and is 10-100 times more than yeast spheroplast transformation.
3. Bacterial colony lift and hybridization techniques can be employed for library screening.
4. F plasmids being circular are east to isolate by mini prep methods.
5. Isolated plasmids can be easily reintroduced.
6. A set of genes for F’ maintenance are sufficient to sustain large fragments of foreign DNA in BAC.
7. A low frequency of co-cloning exists in BACs which confers a significant advantage in physical mapping by ‘bottom up ‘ approach.

Yeast Vectors
2µm plasmid of Yeast:
One of the limited numbers of plasmids found in eukaryotic cells is 2µm plasmid of Saccharomyces cereviieae It size is 6kb.Its copy number is 70-200. Rep 1 and Rep 2 are the two plasmid encoded proteins required for replication along with host proteins. 2µm plasmid also contains flp gene which encodes for a protein that converts A form to B form, in which the order of the genes is rearranged in a intramolecular rearrangement. Another protein encoded by the 2µm plasmid is D protein whose function is unknown.



Genes involved in amino acid biosynthesis are usually used as Selection markers. For instance leu2 encodes for ß- isopropyl malate dehydrogenase, one of the enzymes that converts pyruvate to leucine. leu 2-, an auxotrophic mutant that cannot synthesize leucine and can grow only on medium that is supplemented with leucine. Transformants can grow on minimal media but non transformants cannot grow.

Yeast vectors are mostly developed based on the 2µm plasmid and are usually shuttle vectors.

A shuttle vector is a vector that contains two origins like 2µm plasmid origin and pBR322 sequences, which facilitates the propagation of the vectors in more than organisms.

Yeast episomal plasmids:
Episome is a plasmid with dual existence i.e. it can exist as a plasmid independently or can get integrated into the genome of the host. Integration occurs by homologous recombination between leu 2 regions adjacent to the gene of interest and yeast mutant leu 2- resulting in integration of the whole vector and can get excised back in a similar manner.



Yeast integrative plasmids:
These are basically bacterial plasmids carrying yeast genes, For example YEP5 is a vector with pBR322 background containing URA3 gene from yeast as selection marker, which codes for Orotidine-5’-phosphate decarboxylase, involved in pyrimidine biosynthesis. These vectors cannot replicate independently and has to necessarily integrate into to the host chromosomal DNA for survival as they lack their own origins.

Yeast replicative plasmids:
These vectors contain yeast chromosomal regions containing 2µm plasmid origins and adjacent genes which can act as selection markers. For example YRp7 is a vector with pBR322 background containing 2µm plasmid origins and TRP1 region as selection marker, which codes for genes involved in tryptophan biosynthesis.



YAC vectors: YAC vectors are eukaryotic cloning vectors which contain three essential features. Origins known as Autonomously replicating sequences (ARS), Centromeres and Telomeres. Centromeres are essential for proper segregation of the vector during cell division. Telomeres are essential to prevent loss of DNA sequences present at the ends during DNA replication. YAC vectors have an insert capacity of upto 2MB (2000kb). YAC vectors like BAC vectors are low copy number. vectors. For instance, YAC 3 is a vector with pBR322 background containing 2µm plasmid origins, TRP1 region, URA3 region, CEN 4 and two Tel regions.


Vectors based on Ti plasmid:
Wild type Agrobacterium Ti plasmid cannot be used as gene cloning vectors because of

1. Large size of Ti plasmid; difficult to handle
2. presence of oncogenes or tumor causing genes (auxin and cytokinin)
3. lack of unique restriction sites and marker site within T-DNA

There are two genetically engineered Ti plasmid based vectors. They are Co-integrate vectors and binary vectors.

Co-integrate vectors
In this strategy, both the T-DNA with our gene of interest and vir region are present in the same vector used for transformation. At first; an intermediate vector is made using E.coli plasmid + vir region + T-DNA borders + origin of replication + pBR 322 sequences.

Second vector is a disarmed pTi vector = gene of interest + some markers + pBR 322 sequences

Both intermediate vector and disarmed pTi has some sequences in common (pBR 322 sequences). Therefore by homologous recombination, co-integration of two plasmids will take place within Agrobacterium. Now we have a cointegrate vector that has both T-DNA with our gene of interest with in the T-DNA borders and vir region. This complete vector is used for transformation eg: pGV2260.

Binary vector
In this vector system a rwo vector strategy is employed and hence the name – binary. Here two vectors are used. This vector was devised based on the knowledge that vir region need not be in the same plasmid along with T-DNA for T DNA transfer.

Binary vector consists of a pair of plasmids

1. A disarmed Ti plasmid: This plasmid has T-DNA with gene of interest + ori for both E.coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: pBin 19
2. Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to the plant.

Gateway Vectors:
The Gateway system is designed to surpass the several steps required to perform the cloning in a traditional manner. It adopts a simple two-step procedure sing phage ? site-specific recombinase. Initially as a prerequisite to use the Gateway system, the gene of interest is required to be cloned by conventional means in a Gateway entry vector. This vector carries two att sites that are recognized by the ? site-specific recombinase and the cloned gene should lie between them. Moving this cloned gene to another vector (destination vector) is very simple. The entry vector containing the cloned gene is mixed with the destination vector and ? recombinase in vitro and after a short incubation period the desired recombinant is selected by transformation.

The beauty of the Gateway system is that after the initial entry clone is made the gene of interest can be transferred to many other vectors while maintaining orientation and reading frame with high efficiencies (>99%).

Published date : 21 Jun 2014 05:34PM