PGreen

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The pGreen plasmids are vectors for plant transformation. They were first described in 2000 as components of a novel T-DNA binary system. [1] The supporting web page [2] provides supplementary information and ongoing support to researchers to request their plasmid resources. As these plasmids have been taken up by the research community, the plasmids have been developed, expanding the resources available to the community.

Contents

Researchers are encouraged to contribute to this research community by submitting their vector sequence to genbank and providing a description of the plasmid on the site.

pGreenI and pGreenII

pGreen is the original pGreen plasmid. [3] pGreenII features plasmid backbone modification to improve plasmid stability. [4]

T-DNA regions

No transformation selection

pGreenII 0000: minimal T-DNA with Left and Right border, lacZ gene for blue/white selection during cloning multiple cloning site derived from pBluescript. [5]

pGreenII 62-SK: derived from pGreenII 0000, the LacZ blue/white cloning selection has been replaced with a 35S-MCS-CaMV cassette that allows the insertion of a gene of interest into a 35S over-expression cassette. The multiple cloning site (MCS) is derived from pBluescript. [6] [7]

Kanamycin selection

pGreenII 0029: derived from pGreenII 0000, a nos-kan cassette has been inserted into the HpaI site of the Left Border, providing resistance to kanamycin during plant transformation selection. [8]

pGreenII 0029 62-SK: derived from pGreenII 0029, the LacZ blue/white cloning selection has been replaced with a 35S-MCS-CaMV cassette that allows the insertion of a gene of interest into a 35S over-expression cassette. The MCS is derived from pBluescript. [9]

Hygromycin selection

pGreenII 0179: derived from pGreenII 0000, a 35S-hyg cassette has been inserted into the HpaI site of the Left Border, providing resistance to hygromycin during plant transformation selection. [10]

Bialaphos selection

pGreenII 0229: derived from pGreenII 0000, a nos-bar cassette has been inserted into the HpaI site of the Left Border, providing resistance to bialaphos or phosphinothricin during plant transformation selection. [11] [12]

pGreenII 0229 62-SK: derived from pGreenII 0229, the LacZ blue/white cloning selection has been replaced with a 35S-MCS-CaMV cassette that allows the insertion of a gene of interest into a 35S over-expression cassette. The MCS is derived from pBluescript. [13]

pSoup

This is the helper plasmid that provides the replicase function for the pSa replication origin of pGreen. pSoup is tetracyclin resistant and a complementary incompatibility group such that it can co-exist with pGreen in the Agrobacterium cell.

pSoup: the original help plasmid for pGreen. pGreen will not replicate in Agrobacterium if it is not present. [14]

Related Research Articles

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

<span class="mw-page-title-main">Genetic transformation</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

<i>Agrobacterium tumefaciens</i> Bacterium, genetic engineering tool

Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.

<i>Agrobacterium</i> Genus of bacteria

Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.

<span class="mw-page-title-main">Transfer DNA</span> Type of DNA in bacterial genomes

The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

<span class="mw-page-title-main">Ti plasmid</span> Circular plasmid used in creation of transgenic plants

A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A. vitis.

<span class="mw-page-title-main">Multiple cloning site</span> Dense cluser of restriction sites in DNA

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region.

pBR322 Artificial plasmid

pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher and Raymond L. Rodriguez. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."

<span class="mw-page-title-main">FLP-FRT recombination</span> Site-directed recombination technology

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 μ plasmid of baker's yeast Saccharomyces cerevisiae.

<span class="mw-page-title-main">Blue–white screen</span> DNA screening technique

The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are T-DNA binary vectors and are often replicated in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a plant-virulent bacterium used to insert the recombinant DNA into plants.

A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a T-DNA binary vector and a virhelper plasmid. The two plasmids are used together to produce genetically modified plants. They are artificial vectors that have been derived from the naturally occurring Ti plasmid found in bacterial species of the genus Agrobacterium, such as A. tumefaciens. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

pUC19 Plasmid cloning vector

pUC19 is one of a series of plasmid cloning vectors designed by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix and the abbreviation for the University of California, where early work on the plasmid series had been conducted. The pUC plasmids are all circular double stranded DNA about 2700 base pairs in length. The pUC plasmids are some of the most widely used cloning vectors. This is in part because cells that have successfully been transformed can be easily distinguished from those that have not based on color differences of colonies. pUC18 is similar to pUC19, but the MCS region is reversed.

<span class="mw-page-title-main">Bialaphos</span> Chemical compound

Bialaphos is a natural herbicide produced by the bacteria Streptomyces hygroscopicus and Streptomyces viridochromogenes. It is also known by the ISO common name bilanafos. Bialaphos is a protoxin and nontoxic as is. When it is metabolized by a plant, the glutamic acid analog glufosinate is released which inhibits glutamine synthetase. This results in the accumulation of ammonium and disruption of primary metabolism.

<span class="mw-page-title-main">G-less cassette</span>

The G-less cassette transcription assay is a method used in molecular biology to determine promoter strength in vitro. The technique involves quantification of an mRNA product with the use of a plasmid. The G-less cassette is part of a pre-constructed vector, usually containing a multiple cloning site (MCS) upstream of the cassette. For this reason, promoters of interest can be inserted directly into the MCS to ultimately measure the accuracy and efficiency of a promoter in recruiting transcription machinery.

<span class="mw-page-title-main">Golden Gate Cloning</span> Molecular cloning method for DNA assembly

Golden Gate Cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIS enzymes include BsaI, BsmBI, and BbsI.

References

  1. ↑ Hellens, Roger P.; Edwards, E. Anne; Leyland, Nicola R.; Bean, Samantha; Mullineaux, Philip M. (2000). "PGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation". Plant Molecular Biology. 42 (6): 819–832. doi:10.1023/A:1006496308160. PMID   10890530.
  2. ↑ "www.pGreen.ac.uk". Archived from the original on 2017-07-25. Retrieved 2020-02-13.
  3. ↑ "Cloning Vector pGreen". NCBI Nucleotide. 2006-11-14.
  4. ↑ "Cloning vector pGreenII, complete sequence". NCBI Nucleotide. 2007-05-21.
  5. ↑ "Cloning vector pGreenII 0000, T-DNA region". NCBI Nucleotide. 2007-08-19.
  6. ↑ "Cloning vector pGreenII 62-SK, T-DNA region". NCBI Nucleotide. 2007-08-19.
  7. ↑ "Cloning vector pGreenII 0800, T-DNA region". NCBI Nucleotide. 2007-08-19.
  8. ↑ "Cloning vector pGreenII 0029, T-DNA region". NCBI Nucleotide. 2007-08-19.
  9. ↑ "Cloning vector pGreenII 0029 62-SK, T-DNA region". NCBI Nucleotide. 2007-08-19.
  10. ↑ "Cloning vector pGreenII 0179, T-DNA region". NCBI Nucleotide. 2007-08-19.
  11. ↑ "Bialaphos selection". Toku-E.
  12. ↑ "Cloning vector pGreenII 0229, T-DNA region". NCBI Nucleotide. 2007-08-19.
  13. ↑ "Cloning vector pGreenII 0229 62-SK, T-DNA region". NCBI Nucleotide. 2007-08-19.
  14. ↑ "Cloning vector pSoup, complete sequence". NCBI Nucleotide. 2007-08-19.
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