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Selectable genetic markers are exogenous genes that are introduced into a cell, conferring a previously absent selective advantage. These markers are primarily used to "mark" the successful ligation of DNA into a plasmid and subsequent transformation into a cell. Oftentimes, selectable markers are accompanied by other exogenous genes that is the primary gene of interest; the marker simply serves to distinguish between successful transformations, and unaltered wild-type cells. | Selectable genetic markers are exogenous genes that are introduced into a cell, conferring a previously absent selective advantage. These markers are primarily used to "mark" the successful ligation of DNA into a plasmid and subsequent transformation into a cell. Oftentimes, selectable markers are accompanied by other exogenous genes that is the primary gene of interest; the marker simply serves to distinguish between successful transformations, and unaltered wild-type cells. | ||
It is not atypical to witness transformation efficiencies as low as 0.05%, making it difficult to pick correct cellular colonies without additional techniques. This is where the selectable genetic markers prove their usefulness. For instance, selectable genetic markers can be used to confer ampicillin resistance to E. coli. These newly resistant E. coli can then be grown on culture plates with ampicillin, allowing only E.coli with successfully transformed DNA to proliferate. | It is not atypical to witness transformation efficiencies as low as 0.05%, making it difficult to pick correct cellular colonies without additional techniques. This is where the selectable genetic markers prove their usefulness. For instance, selectable genetic markers can be used to confer ampicillin resistance to <i>E. coli</i>. These newly resistant <i>E. coli</i> can then be grown on culture plates with ampicillin, allowing only <i>E.coli</i> with successfully transformed DNA to proliferate. | ||
In addition to selectable genetic markers are screenable genetic markers. Screenable genetic markers function in a similar manner in that they are exogenous genes that are transformed into a cell; however, they do not confer any new sort of resistance to the cell. Instead, they cause the cell to respond differently to environmental conditions in such a way as to distinguish transformed cells from untransformed cells. This can be useful when determining the transformation efficiency of a cell, or when carefully monitoring the activity of proteins. | In addition to selectable genetic markers are screenable genetic markers. Screenable genetic markers function in a similar manner in that they are exogenous genes that are transformed into a cell; however, they do not confer any new sort of resistance to the cell. Instead, they cause the cell to respond differently to environmental conditions in such a way as to distinguish transformed cells from untransformed cells. This can be useful when determining the transformation efficiency of a cell, or when carefully monitoring the activity of proteins. | ||
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===Antibiotic=== | ===Antibiotic=== | ||
In synthetic biology research, the primary forms of selectable markers are antibiotic resistant genes. Because a large portion of research takes place in vivo in E. coli, antibiotic selectable markers can be employed whenever transfecting DNA in order to distinguish wild-type cells from successfully transfected ones. If ligating more than one gene of interest into a plasmid for transfection into E. coli, it is often beneficial to employ multiple antibiotic markers to ensure that both genes are present in resultant colonies. | In synthetic biology research, the primary forms of selectable markers are antibiotic resistant genes. Because a large portion of research takes place in vivo in <i>E. coli</i>, antibiotic selectable markers can be employed whenever transfecting DNA in order to distinguish wild-type cells from successfully transfected ones. If ligating more than one gene of interest into a plasmid for transfection into <i>E. coli</i>, it is often beneficial to employ multiple antibiotic markers to ensure that both genes are present in resultant colonies. | ||
Common types of antibiotics used include ampicillin, tetracycline, chloramphenicol, and the many -mycins, including kanamycin. A large range of antibiotic resistances are used as genetic markers. Because of this, each antibiotic resistance is often referred to by a three letter acronym, such as Amp, Tet, Chl, Cam and Kan. Plates containing these antibiotics can be made en mass, and used to grow appropriate cultures of transformed E. coli. | Common types of antibiotics used include ampicillin, tetracycline, chloramphenicol, and the many -mycins, including kanamycin. A large range of antibiotic resistances are used as genetic markers. Because of this, each antibiotic resistance is often referred to by a three letter acronym, such as Amp, Tet, Chl, Cam and Kan. Plates containing these antibiotics can be made en mass, and used to grow appropriate cultures of transformed <i>E. coli</i>. | ||
Antibiotic markers are the most popular form of selectable genetic markers. As such, the field is quite large and constantly expanding in order to meet research needs. For instance, <i>Poggi et al.</i> recognized the mutation of antibiotic resistance towards gentamicin, kanamycin, streptomycin, and spectinomycin in leptospiral pathogens. The group was able to develop a cassette that included two antibiotic markers, along with a new gentamicin marker. Using multiple antibiotic markers greatly reduces the chance of background colonies that have spontaneously developed antibiotic resistance<cite>Poggi2010</cite>. | Antibiotic markers are the most popular form of selectable genetic markers. As such, the field is quite large and constantly expanding in order to meet research needs. For instance, <i>Poggi et al.</i> recognized the mutation of antibiotic resistance towards gentamicin, kanamycin, streptomycin, and spectinomycin in leptospiral pathogens. The group was able to develop a cassette that included two antibiotic markers, along with a new gentamicin marker. Using multiple antibiotic markers greatly reduces the chance of background colonies that have spontaneously developed antibiotic resistance<cite>Poggi2010</cite>. | ||
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===Herbicidal=== | ===Herbicidal=== | ||
Oftentimes, researchers find themselves working not with E. coli or other bacteria, but with plant organisms that are unaffected by antibiotics. In these instances, antibiotic resistance is replaced with herbicidal resistance. While the overall process remains essentially the same, herbicide resistance falls under a different category of selectable genetic markers. | Oftentimes, researchers find themselves working not with <i>E. coli</i> or other bacteria, but with plant organisms that are unaffected by antibiotics. In these instances, antibiotic resistance is replaced with herbicidal resistance. While the overall process remains essentially the same, herbicide resistance falls under a different category of selectable genetic markers. | ||
One of the most common forms of herbicide resistance found in the world is glyphosate resistance. Glyphosate, a common herbicide found especially in Roundup, is a competitor of the enzyme 5-enolpyruvoyl-shikimate-3-phosphate synthetase (EPSPS). The herbicide acts as a transition state analog, binding readily to EPSPS and thus inhibiting the Shikimate pathway. Monsanto introduced glyphosate resistance by first isolating a variant of EPSPS from Agrobacterium (a gram-negative bacteria) strain CP4 in the 1980s, with the unique feature of not being inhibited by glyphosate<cite>Funke2006</cite>. The Monsanto corporation introduced glyphosate resistance into soybeans in 1996, and provides an excellent example of the commercial application of selectable genetic markers. Since then, Monsanto has incorporated glyphosate resistance into other plants such as canola, corn, and alfalfa. Approximately 50% of all agricultural land in the United States is now occupied by these variants, attesting to the power of selectable genetic markers<cite>Owen2010</cite>. | One of the most common forms of herbicide resistance found in the world is glyphosate resistance. Glyphosate, a common herbicide found especially in Roundup, is a competitor of the enzyme 5-enolpyruvoyl-shikimate-3-phosphate synthetase (EPSPS). The herbicide acts as a transition state analog, binding readily to EPSPS and thus inhibiting the Shikimate pathway. Monsanto introduced glyphosate resistance by first isolating a variant of EPSPS from Agrobacterium (a gram-negative bacteria) strain CP4 in the 1980s, with the unique feature of not being inhibited by glyphosate<cite>Funke2006</cite>. The Monsanto corporation introduced glyphosate resistance into soybeans in 1996, and provides an excellent example of the commercial application of selectable genetic markers. Since then, Monsanto has incorporated glyphosate resistance into other plants such as canola, corn, and alfalfa. Approximately 50% of all agricultural land in the United States is now occupied by these variants, attesting to the power of selectable genetic markers<cite>Owen2010</cite>. | ||
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Recent research into selectable genetic markers has looked into pathways that avoid employing antibiotic and herbicidal resistance. This is due to rising concern over "wild" strains of bacteria or plants developing antibiotic or herbicidal resistance and proliferating rapidly in nature. Even in a laboratory environment, avoiding the resistance approach towards selectable markers can prove beneficial. | Recent research into selectable genetic markers has looked into pathways that avoid employing antibiotic and herbicidal resistance. This is due to rising concern over "wild" strains of bacteria or plants developing antibiotic or herbicidal resistance and proliferating rapidly in nature. Even in a laboratory environment, avoiding the resistance approach towards selectable markers can prove beneficial. | ||
A novel approach towards selectable markers was developed in Lawrence Livermore National Laboratory, which employes a toxin/antitoxin combination of genes as a marker. The process, summarized in the figure to the left, effectively avoids the need to grow antibiotic resistant bacterial cultures on an antibiotic plate. An inducible zeta-toxin group of proteins is first introduced into an E. coli strain. A DNA strand of interest containing an zeta-antitoxin group is then transformed into the E. coli, and the entire culture is grown. The zeta-toxin group is then induced, killing off all E. coli that does not contain the antitoxin group. Besides for triggering the zeta-toxin group, no outside influence is required to select for the desired cells<cite>Parsons2011</cite>. | A novel approach towards selectable markers was developed in Lawrence Livermore National Laboratory, which employes a toxin/antitoxin combination of genes as a marker. The process, summarized in the figure to the left, effectively avoids the need to grow antibiotic resistant bacterial cultures on an antibiotic plate. An inducible zeta-toxin group of proteins is first introduced into an <i>E. coli</i> strain. A DNA strand of interest containing an zeta-antitoxin group is then transformed into the <i>E. coli</i>, and the entire culture is grown. The zeta-toxin group is then induced, killing off all <i>E. coli</i> that does not contain the antitoxin group. Besides for triggering the zeta-toxin group, no outside influence is required to select for the desired cells<cite>Parsons2011</cite>. | ||
To read more about toxin/antitoxin systems, [http://openwetware.org/wiki/CH391L/S12/ToxinAntitoxins see this page]. Additionally, read about [http://openwetware.org/wiki/CH391L/S12/CounterSelection counter-selective markers], as an alternative to selective markers. | To read more about toxin/antitoxin systems, [http://openwetware.org/wiki/CH391L/S12/ToxinAntitoxins see this page]. Additionally, read about [http://openwetware.org/wiki/CH391L/S12/CounterSelection counter-selective markers], as an alternative to selective markers. | ||
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===Blue/White Screening=== | ===Blue/White Screening=== | ||
Blue/White Screening is commonly used in E. coli transformations. In this screening, cells are grown on agar plates in the presence of X-gal and IPTG to test for the presence of β-galactosidase enzyme. In the M15 strain of E. coli, part of the <i>lacZ</i> gene is deleted, removing the cell's ability to produce β-galactosidase. However, when transfected with a plasmid containing a <i>lacZα</i> domain, such as pUC19, the gene becomes operable and the cell produces β-galactosidase. It is possible to create a successful transformation in which β-galactosidase is not produced by inserting DNA into the <i>lacZα</i> domain. This is particularly useful to check for successful ligations. Successful ligations will not produce β-galactosidase, while unsuccessful ligations will. | Blue/White Screening is commonly used in <i>E. coli</i> transformations. In this screening, cells are grown on agar plates in the presence of X-gal and IPTG to test for the presence of β-galactosidase enzyme. In the M15 strain of <i>E. coli</i>, part of the <i>lacZ</i> gene is deleted, removing the cell's ability to produce β-galactosidase. However, when transfected with a plasmid containing a <i>lacZα</i> domain, such as pUC19, the gene becomes operable and the cell produces β-galactosidase. It is possible to create a successful transformation in which β-galactosidase is not produced by inserting DNA into the <i>lacZα</i> domain. This is particularly useful to check for successful ligations. Successful ligations will not produce β-galactosidase, while unsuccessful ligations will. | ||
X-gal, while normally colorless (i.e. white), will readily hydrolyze in the presence of β-galactosidase into a compound with a sharp blue color. Therefore, colonies with successfully transformed cells with the desired DNA will grow white, while background colonies will grow blue. | X-gal, while normally colorless (i.e. white), will readily hydrolyze in the presence of β-galactosidase into a compound with a sharp blue color. Therefore, colonies with successfully transformed cells with the desired DNA will grow white, while background colonies will grow blue. | ||
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#Reinthaler2002 pmid=12697213 | #Reinthaler2002 pmid=12697213 | ||
//Development of antibiotic resistance in wild-type E. coli. | //Development of antibiotic resistance in wild-type <i>E. coli</i>. | ||
</biblio> | </biblio> | ||
