Difference between revisions of "CH391L/S14/Ashley's First assignment"

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[[Category:CH391L_S12]]
 
[[Category:CH391L_S12]]
  
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Ashley Kessel's first assignment. Cloned wiki.
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Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)
  
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==Counterselectable Genetic Markers==
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==Selectable Markers Overview==
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[[Image:Antibiotic Resistance Markers.jpg‎|thumb|right|Example application of selectable genetic markers in nematodes. Only nematodes transfected with markers survive and proliferate<cite>Giordano-Santini2011</cite>.]]
  
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===Introduction===
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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.  
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In contrast to selection markers, counter-selection markers serve to eliminate unwanted elements. These markers are often toxic or otherwise inhibitory to replication under certain conditions.  Selective conditions often involve exposure to a specific substrates or shift in growth conditions. These elements are often incorporated into genetic modification schemes in order to select for rare recombination events that require the removal of the marker or to selectively eliminate plasmids or cells from a given population.
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===Application: Allelic Exchange===
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[[Image:SacB Allelic.jpg| Allelic Exchange. Marx C.J. BMC Research Notes. 2008| right |thumb|200px]]
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The introduction of specific mutations in a genetic sequence is a powerful way to learn about gene function or to engineer an organism for a desired application.  A common way to introduce specific mutations into a target sequence is through allelic exchange. In a typical allelic exchange experiment, the chromosomal sequence to be mutated is synthesized and cloned onto a vector. This sequence is either highly homologous (except for the introduced mutations) to the chromosomal version or else is flanked by homologous sequences specifying the desired insertion site. Upon introduction to the cell, the cell’s homologous recombination machinery will recognize the sites of homology between the vector and the chromosome and at some frequency will stimulate the integration of the vector at the site of homology.  This event is often selected for by the presence of a selectable marker present on the vector.  Following this selection, it is often desirable to remove the vector to produce a “seemless” insertion. For this purpose, a counterselectable marker is included on the vector.  Upon induction of the counterselectable condition, only those cells that have excised the vector sequence through a second recombination event will survive. Since this recombination event is usually rare the counterselection step is essential to find the desired mutants.
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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.
  
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===Application: Plasmid curing===
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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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It is often necessary to "cure" or isolate cells that have lost a plasmid.  This can typically be achieved through serial passage in non-selective media and screening for plasmid loss.  Counterselective markers can be incorporated onto plasmids so that their loss can be selected for.  A common strategy includes the use of a temperature sensitive origin of replication.  These engineered origins permit replication at a permissive temperature but prevent replication upon switch to the counterselective temperature.  After a period of growth at the selective temperature, the plasmid will be lost because it fails to replicate when the cell divides.  Another strategy involves the inclusion of a counterselectable marker (see "parts" below) on the vector allowing for selection of cells that lose the plasmid on their own.
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==Types of Selectable Markers==
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=='''Parts'''==
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====tetAR====
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===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 <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.
  
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The tetAR genes endow the cell with resistance to the antibiotic tetracycline by altering the cell membrane making it impermeable to the drug.  However, this alteration makes the cell hypersensitive to lipophilic chelating agents such as fusaric or quinalic acids. This enables selection of cells that have lost the tetAR genes by exposure to fusaric acid<cite>Bochner1980</cite>.
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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>.
  
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====sacB====
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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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sacB is perhaps the most widely used counterselectable marker.  The sacB gene was isolated from ''Bacillus subtilis'' and encodes the for the enzyme levansucrase.  Expression of sacB in most gram-positive bacteria is harmless, but is lethal when expressed in gram-negative bacteria in the presence of sucrose <cite>Gay1985</cite>. The mechanism of toxicity is not completely understood but it is believed to be caused by accumulation of levans (high molecular weight fructose polymers) in the periplasm of gram-negatives.
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The evolution of antibiotic tolerance and eventual resistance in laboratory bacteria is a potential issue when performing experiments with antibiotic selective markers. A study conducted on <i>E. coli</i> in the effluent of waste-water treatment plants, which employ numerous antibiotics, found antibiotic resistance in 16 of the 24 antibiotics tested<cite>Reinthaler2002</cite>. The researchers also found that effluent from areas that employed antibiotics more frequently, such as hospitals, contained <i>E. coli</i> with proportionally more antibiotic resistance. Resistance to the [http://en.wikipedia.org/wiki/Penicillin penicillin], [http://en.wikipedia.org/wiki/Cephalosporin cephalosporin], and [http://en.wikipedia.org/wiki/Quinolone quinolones], group of antibiotics was especially prevalent, as well as resistance to [http://en.wikipedia.org/wiki/Sulfamethoxazole sulfamethoxazole] and [http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0000576/ tetracycline] <cite>Reinthaler2002</cite>.
  
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====rpsL====
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===Herbicidal===
 +
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.
  
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The rpsL gene encodes the S12 protein component of the 30S ribosome. Certain mutations of the gene cause resistance to the antibiotic streptomycin which targets the 30S ribosome. Resistance to streptomycin resistance is recessive meaning that an additional wild-type copy of the gene will lead to streptomycin sensitivity<cite>Lederberg1951</cite>. Therefore, the wild-type rpsL gene can be  used as a counterselective marker in a strain that already possesses a mutant allele. Selection on streptomycin will only permit those cells that have lost the wild-type gene to survive.
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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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====ccdB====
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Selectable genetic markers for plants are not always in the form of herbicide resistance. For instance, researchers at China's Agricultural University were able to express the rstB gene in tobacco, which confers upon the plant greater tolerance to salt concentration. <i>Zhang et al.</i> was able to achieve approximately 80% selection efficiency using salt concentrations at 170mM, proving remarkable success in using a selectable genetic marker other than herbicide resistance<cite>Zhang2008</cite>.
  
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[[Image:Gateway_cloning.gif‎ | Invitrogen "Gateway" cloning system| right |thumb|200px]]
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===Auxotrophic===
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ccdB is the toxin component of the toxin-antitoxin system of the F plasmid.  ccdB is a DNA gyrase inhibitor which causes cell death when the ccdA antitoxin is not present.  There is a known mutation of the DNA gyrase gene (gyrA462) that confers resistance to the ccdB toxin<cite>Bernard1992</cite>.  This allows plasmid vectors that contain ccdB to be propagated without associated toxicity in the absence of ccdA  Cloning vectors that contain the ccdB gene can be used to select against vectors that fail to accept a desired insert when transformed into a wild-type gyrA strain.  This cloning scheme virtually eliminates background.  The Invitrogen "Gateway" cloning system takes advantage of this method [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html].
+
  
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====URA3====
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Auxotrophy is a condition in which an organism cannot produce the compounds necessary for its survival. For instance, humans cannot naturally produce vitamins, and must obtain them from their diet. Auxotrophy can be applied in a laboratory setting by altering an organism so as to remove its ability to produce a necessary compound. For instance, one could employ PCR mediated gene disruption to insert a gene in such a way as to inhibit a gene, crucial for the production of organic compounds, from functioning. This can create a strain of bacteria, yeast, or other easily manipulable organism that is incapable of producing the basic compounds for its survival. However, when supplied with these compounds from their environment, these organisms maintain the ability to survive. This is useful as it creates organisms that will die of their own accord, unless specifically kept alive by experimenters. One can then transform a plasmid into these organisms with a gene that allows for the production of this compound, and grow the organism in media without this compound. Only successfully transformed organisms will survive in this environment.
  
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The URA3 gene from ''Saccharomyces cerevisiae'' encodes for orotidine-5'-monophosphate decarboxylase which is involved in de novo synthesis of pyrimidine nucleotides (pathway [http://pathway.yeastgenome.org/YEAST/NEW-IMAGE?type=PATHWAY&object=PYRIMID-RNTSYN-PWY&detail-level=2]).  URA3 normally catalyzes the decarboxylation of orotidine 5-phosphate (OMP) to uridylic acid (UMP).  The gene is especially useful as a marker in that it can be used for both selection and counterselection<cite>Boeke1984</cite>.  Many common yeast strains contain a mutation in the URA3 gene making the cells auxotrophic for Uracil.  Supplying an intact copy of the URA3 gene on a plasmid or integration cassette restores prototrophy and can be used to select for cells which have taken up the vector containing the marker.  URA3 is also known to convert 5-Fluoroorotic acid (5-FOA) into the toxic compound 5-fluorouracil leading to cell death.  Therefore, counterselection on 5-FOA will select for clones which have lost the URA3 marker.
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Auxotrophic selective markers are commonly used in experiments involving yeast strains. For a list of common auxotrophic selective markers used in yeast, [http://www.yeastgenome.org/community/alleletable.shtml look here].
  
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===Other===
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[[Image:Alternative Selective Marker.jpg|thumb|left|An alternative technique for selectable markers that avoids antibiotic resistance<cite>Parsons2011</cite>.]]
  
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It is important to note that the URA3/5-FOA system only works in ''Saccharomyces cerevisiae'' in strains with a chromosomal URA3 mutation.  Recently the system has been ported to bacterial species<cite>Galvao2005</cite>.  By identifying the URA3 homolog, (pyrF in ''E.coli'' and ''p.putida'') and deleting it, URA3 selection/counterseletion can be employed.
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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.
  
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====pheS====
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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>.
  
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pheS encodes the α subunit of the Phenylalanine-tRNA sythetase. A known mutation in this protein (G294A) relaxes the substrate specificity of the enzyme causing toxic phenylalanine analogs like p-chlorophenylalanine to be incorporated into proteins in place of phenylalanine<cite>Kast1992</cite>. When grown on media containing p-chlorophenylalanine, cells harboring the mutant version of the gene will be eliminated. Unlike the rpsL counterselection, the mutants pheS phenotype is dominant meaning that the presence of the mutant gene will still cause toxicity when the wild-type version is also present.  This strategy has been used in both Gram-negative and Gram-positive bacteria.
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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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====Thymidine Kinase====
 
  
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Thymidine kinase (TK) is a popular counterselection tool used in mammalian cell culture. TK is involved in the salvaging of nucleotides for DNA synthesis through the phosphorylation of deoxythymidine to deoxythymidine 5'-phosphate (TMP). Cells lacking TK will not grow on media that blocks the de novo synthesis pathway. For this reason HAT (Hypoxanthine-aminopterin-thymidine) media can be used to select for TK knockout cells that contain an introduced copy of TK.
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==Types of Screening==
 +
[[Image:Blue white test.jpg|thumb|right|Successful example of a blue/white screen test. Blue colonies are wild-type cells, while white colonies are successfully transformed cells.]]
  
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If a given cell line is deficient for TK, growth on HAT media which contains aminopterin will prevent the production of tetrahydrofolate (THF) through the inhibition of dihydrofolate reductase. THF is a required cofactor needed for the de novo production of TMP by Thymidylate synthase. With the de novo pathway disabled, the cell will need to have a functional copy of TK to phosphorylate the available Thymidine in the media to TMP and resume DNA synthesis.
+
===Blue/White Screening===
 +
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.
  
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Cells containing the Thymidine kinase gene can then be counterselected against by the addition of toxic Thymidine analogs such as 5-bromo-deoxyuridine or Ganciclovir. These Thymidine analogs are chain terminators which do not have a hydroxyl group in the 3'-position which is required for continued chain elongation. Thus, cells with TK will be eliminated due to their inability to synthesize complete DNA sequences.
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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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=='''Nutritional'''==
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===Green Fluorescent Protein Screening===
 +
[[Image:Green_Fluroescent_Mice.jpg‎|thumb|left|Mice transfected with GFP. One can easily distinguish the wild-type mouse (middle) from the two mice with GFP (left and right)<cite>Moen2011</cite>.]]
  
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Counterselection against certain populations of cells can also be obtained through the use of strains auxotropic for a particular metabolite.  A point mutation or deletion in a gene required for amino acid synthesis, carbon source metabolism, etc can be used to select against strains when grown on media lacking the required nutrient.  In most cases a defined "minimal" media is required for counterseletion. There are a number of counterselective auxotropic markers that can be used in rich media, examples from ''E. coli'' include ''thyA'' and ''dapA-E''.
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Green Fluorescent Protein, or GFP, was first isolated from the crystal jellyfish Aequorea victoria in the 1960s. In 1994, GFP was successfully cloned<cite>Chalfie1994</cite>, allowing researchers to use the protein as a screenable marker for the first time. Virtually harmless in live cells, GFP has the unique phenotype of glowing bright green under ultraviolet light. GFP functions entirely of its own accord, and requires no exogenous material besides ionizing radiation in order to fluoresce. This allows GFP to be used as a marker accompanying transfected DNA, and has been used extensively in academia.
  
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ThyA encodes thymidylate synthetase which is involved in de novo synthesis of dTMP from dUMP which is required for DNA synthesis [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=PWY0-166&detail-level=2]. A mutation in ''thyA'' makes the cell severely auxotrophic for Thymine. A useful aspect of this marker is that growth in rich media does not provide enough Thymine to support growth of ''thyA'' mutants, enabling selection against the ''thyA'' mutant. Similarly a mutation in the gene ''dapA'' which encodes Dihydrodipicolinate synthase makes the cell auxotrophic even in rich media for diaminopimelic acid which is required for Lysine and cell wall (peptidoglycan) biosynthesis [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=DAPLYSINESYN-PWY].
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The scientist to first discover GFP, [http://www.npr.org/templates/story/story.php?storyId=95545761 Douglas Prasher], was not included in the eventual Nobel prize it was associated with in 2008. Prasher had run out of funding for his research in the 1990s at Woods Hole Oceanographic Institution after donating his isolated GFP gene to Tsien and Chalfie, two of the scientists who eventually won the Nobel prize for GFP. After his last project proposal was rejected by National Institutes of Health, Prasher moved on to multiple jobs before eventually settling down as a shuttle bus driver for many years. Prasher now works as a senior scientist at [http://www.linkedin.com/pub/douglas-prasher/5/b92/a80 Streamline Automation].
 +
 
 +
In 2011, GFP was used to create an in vivo mammary model to investigate tumorigenesis in mice. Tumor cells were introduced into the mice, accompanied with GFP as a screenable marker. As the mice tumors proliferated, so did GFP. This allowed for easy differentiate between tumors and stroma cells, greatly aiding cancer researchers<cite>Moen2011</cite>.
 +
 
 +
Selectable genetic markers can also be used to investigate protein activity. Research performed at the University of Washington used GFP coupled with inteins to splice GFP into other proteins with greater than 96% efficiency. The GFP proved harmless to the intein's activity, and also allowed the researchers to analyze the effectiveness of the inteins themselves<cite>Ramsden2011</cite>.
 +
 
 +
 
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==Artificial Selection==
 +
Selectable markers have a remarkably relevant role in industrial applications. Because of their ability to distinguish cells from one another, selectable markers are an essential tool for artificial selection. While artificial selection of organisms is possible without the use of selectable markers, the process is significantly shorter with their use.
 +
 
 +
Artificial selection is a special instance in which selectable markers are often the desired gene to be introduced into a cell. For instance, rice has been transfected with a plethora of resistances using selectable markers. Glycopeptide binding protein, dihydrofolate reductase, and hygromycin phosphotransferase have all been introduced into rice, conferring resistance to bleomycin and pheomycin, methotrexate, and hygromycin B respectively. This allows farmers to use herbicides select for only rice with these markers, while eliminating the majority of invasive species<cite>Twyman2002</cite>.
 +
 
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==Issues==
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As mentioned [[#Other|earlier]], current research into genetic markers involves looking at alternatives to antibiotic and herbicidal resistance. Both the commercial and research sectors have reason to adopt alternative techniques. In the commercial sector, there is the omnipresent public fear of growing and consuming "genetically modified crops". In the research sector, new techniques can decrease costs, time, labor, and error rates (it is always possible that wild-type cultures can grow under antibiotic/herbicidal conditions). Either sector would also be held responsible by governments should their modified organisms escape into the environment and proliferate.
 +
===Example: Proliferation of glyphosate resistance===
 +
[[Image:Spread of Glyphosate Resistance.jpg‎|thumb|right|Since the introduction of glyphosate resistance in 1996, the amount of wild weeds resistant to glyphosate has exploded<cite>Owen2010</cite>.]]
 +
 
 +
Since Monsanto's introduction of glyphosate resistance in 1996, there has been a growing concern over the affects of its widespread use. Glyphosate resistance is used primarily in Monsanto's Roundup Ready™ plants to work in tandem with Monsanto's Roundup™ herbicide, whose key active ingredient is glyphosate. Roundup has been used commercially since 1976. However, Roundup's true success did not truly begin until the development of Roundup Ready selective genetic markers.  
 +
 
 +
It was at this time that an explosion in glyphosate resistant weeds began in the environment. Originally, there were no plants natively resistant to glyphosate. Even in 1996, 30 years after the introduction of Roundup, only 2 weeds worldwide had developed glyphosate resistance. However, as shown in the figure to the right, this number increased to 19 weeds by 2010<cite>Owen2010</cite>. The correlation between the introduction of commercial glyphosate resistance and the mutation of invasive weeds is substantial, and raised public concerns.
 +
 
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In 2006, after hearing a case brought against Monsanto by a coalition of farmers and environmental groups, the US District Court of Northern California ruled against Monsanto and issued an injunction on all Roundup Ready alfalfa sales. An appeal to the Ninth Circuit Court of Appeals upheld the District Court's ruling. It was not until a [http://www.monsanto.com/newsviews/Documents/rralfalfa_supreme_court_decision.pdf Supreme Court] hearing in January of 2010 that the decision was reversed and the injunction lifted.
  
 
==References==
 
==References==
 
<biblio>
 
<biblio>
−
#Bochner1980 pmid=6259126
+
#Giordano-Santini2011 pmid=21431833
−
#Gay1985 pmid=2997137
+
//Review article about selectable genetic markers as used in nematodes. Relatively new field for nematodes, possible due to the completion of the <i>Caenorhabditis elegans</i> genome.
−
#Lederberg1951 pmid=14832197
+
 
−
#Bernard1992 pmid=1324324
+
#Moen2011 pmid=22251838
−
#Kast1992 pmid=8125286
+
//Observes tumor growth in mice by introducing GFP into the mice.
−
#Boeke1984 pmid=6394957
+
 
−
#Galvao2005 pmid=15691944
+
#Chalfie1994 pmid=8303295
 +
//The first instance of using GFP as a marker.
 +
 
 +
#Twyman2002 [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=0CDoQFjAC&url=http%3A%2F%2Fwww.springer.com%2Fcda%2Fcontent%2Fdocument%2Fcda_downloaddocument%2F9783540431534-c1.pdf%3FSGWID%3D0-0-45-83701-p2219862&ei=NeRBT9ThF4Wg2gWfvZ2DCA&usg=AFQjCNHO6UlmaFGSkZFpXAeTVSSZX9I1JA Twyman RM, Stroger E, Kohli A, Capell T and Christou P]
 +
//Various genetic markers used for artificial selection of rice crops.
 +
 
 +
#Parsons2011 [https://e-reports-ext.llnl.gov/pdf/476269.pdf D. Parsons, M. Tolmasky, P. Chain and B. W. Segelke]
 +
//Report by the Lawrence Livermore National Laboratory on a new system for selectable markers
 +
 
 +
#Poggi2010 pmid=20511419
 +
//Multiple antibiotic resistances as selectable genetic markers in Leptospira.
 +
 
 +
#Goodwin2005 pmid=15310922
 +
//Herbicide selectable genetic markers in wheat.
 +
 
 +
#Owen2010 [http://www7.nationalacademies.org/ocga/testimony/t_Herbicide_Resistant_Weeds_in_GE_Crops.asp Owen, M.]
 +
//Report by Professor Micheal Owen from Iowa State University on glyphosate resistance for the U.S. House of representatives
 +
 
 +
#Zhang2008 [http://www.springerlink.com/content/u2172658q3p50727/ Zhang WJ, Yang SS, Shen XY, Jin YS, Zhao HJ and Wang T]
 +
//Salt tolerance as a selectable genetic marker.
 +
 
 +
#Ramsden2011 pmid=21708017
 +
//Use of inteins with GFP to introduce selectable markers into proteins.
 +
 
 +
#Funke2006 pmid=16916934
 +
//Molecular basis of glyphosate resistance.
 +
 
 +
#Reinthaler2002 pmid=12697213
 +
//Development of antibiotic resistance in wild-type <i>E. coli</i>.
 +
</biblio>

Revision as of 21:28, 24 January 2014


Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)

Contents

Selectable Markers Overview

File:Antibiotic Resistance Markers.jpg
Example application of selectable genetic markers in nematodes. Only nematodes transfected with markers survive and proliferate[1].

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.

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.

Types of Selectable Markers

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.

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.

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, Poggi et al. 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[2].

The evolution of antibiotic tolerance and eventual resistance in laboratory bacteria is a potential issue when performing experiments with antibiotic selective markers. A study conducted on E. coli in the effluent of waste-water treatment plants, which employ numerous antibiotics, found antibiotic resistance in 16 of the 24 antibiotics tested[3]. The researchers also found that effluent from areas that employed antibiotics more frequently, such as hospitals, contained E. coli with proportionally more antibiotic resistance. Resistance to the penicillin, cephalosporin, and quinolones, group of antibiotics was especially prevalent, as well as resistance to sulfamethoxazole and tetracycline [3].

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.

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[4]. 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[5].

Selectable genetic markers for plants are not always in the form of herbicide resistance. For instance, researchers at China's Agricultural University were able to express the rstB gene in tobacco, which confers upon the plant greater tolerance to salt concentration. Zhang et al. was able to achieve approximately 80% selection efficiency using salt concentrations at 170mM, proving remarkable success in using a selectable genetic marker other than herbicide resistance[6].

Auxotrophic

Auxotrophy is a condition in which an organism cannot produce the compounds necessary for its survival. For instance, humans cannot naturally produce vitamins, and must obtain them from their diet. Auxotrophy can be applied in a laboratory setting by altering an organism so as to remove its ability to produce a necessary compound. For instance, one could employ PCR mediated gene disruption to insert a gene in such a way as to inhibit a gene, crucial for the production of organic compounds, from functioning. This can create a strain of bacteria, yeast, or other easily manipulable organism that is incapable of producing the basic compounds for its survival. However, when supplied with these compounds from their environment, these organisms maintain the ability to survive. This is useful as it creates organisms that will die of their own accord, unless specifically kept alive by experimenters. One can then transform a plasmid into these organisms with a gene that allows for the production of this compound, and grow the organism in media without this compound. Only successfully transformed organisms will survive in this environment.

Auxotrophic selective markers are commonly used in experiments involving yeast strains. For a list of common auxotrophic selective markers used in yeast, look here.

Other

File:Alternative Selective Marker.jpg
An alternative technique for selectable markers that avoids antibiotic resistance[7].

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[7].

To read more about toxin/antitoxin systems, see this page. Additionally, read about counter-selective markers, as an alternative to selective markers.


Types of Screening

File:Blue white test.jpg
Successful example of a blue/white screen test. Blue colonies are wild-type cells, while white colonies are successfully transformed cells.

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 lacZ gene is deleted, removing the cell's ability to produce β-galactosidase. However, when transfected with a plasmid containing a lacZα 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 lacZα 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.


Green Fluorescent Protein Screening

File:Green Fluroescent Mice.jpg
Mice transfected with GFP. One can easily distinguish the wild-type mouse (middle) from the two mice with GFP (left and right)[8].

Green Fluorescent Protein, or GFP, was first isolated from the crystal jellyfish Aequorea victoria in the 1960s. In 1994, GFP was successfully cloned[9], allowing researchers to use the protein as a screenable marker for the first time. Virtually harmless in live cells, GFP has the unique phenotype of glowing bright green under ultraviolet light. GFP functions entirely of its own accord, and requires no exogenous material besides ionizing radiation in order to fluoresce. This allows GFP to be used as a marker accompanying transfected DNA, and has been used extensively in academia.

The scientist to first discover GFP, Douglas Prasher, was not included in the eventual Nobel prize it was associated with in 2008. Prasher had run out of funding for his research in the 1990s at Woods Hole Oceanographic Institution after donating his isolated GFP gene to Tsien and Chalfie, two of the scientists who eventually won the Nobel prize for GFP. After his last project proposal was rejected by National Institutes of Health, Prasher moved on to multiple jobs before eventually settling down as a shuttle bus driver for many years. Prasher now works as a senior scientist at Streamline Automation.

In 2011, GFP was used to create an in vivo mammary model to investigate tumorigenesis in mice. Tumor cells were introduced into the mice, accompanied with GFP as a screenable marker. As the mice tumors proliferated, so did GFP. This allowed for easy differentiate between tumors and stroma cells, greatly aiding cancer researchers[8].

Selectable genetic markers can also be used to investigate protein activity. Research performed at the University of Washington used GFP coupled with inteins to splice GFP into other proteins with greater than 96% efficiency. The GFP proved harmless to the intein's activity, and also allowed the researchers to analyze the effectiveness of the inteins themselves[10].


Artificial Selection

Selectable markers have a remarkably relevant role in industrial applications. Because of their ability to distinguish cells from one another, selectable markers are an essential tool for artificial selection. While artificial selection of organisms is possible without the use of selectable markers, the process is significantly shorter with their use.

Artificial selection is a special instance in which selectable markers are often the desired gene to be introduced into a cell. For instance, rice has been transfected with a plethora of resistances using selectable markers. Glycopeptide binding protein, dihydrofolate reductase, and hygromycin phosphotransferase have all been introduced into rice, conferring resistance to bleomycin and pheomycin, methotrexate, and hygromycin B respectively. This allows farmers to use herbicides select for only rice with these markers, while eliminating the majority of invasive species[11].


Issues

As mentioned earlier, current research into genetic markers involves looking at alternatives to antibiotic and herbicidal resistance. Both the commercial and research sectors have reason to adopt alternative techniques. In the commercial sector, there is the omnipresent public fear of growing and consuming "genetically modified crops". In the research sector, new techniques can decrease costs, time, labor, and error rates (it is always possible that wild-type cultures can grow under antibiotic/herbicidal conditions). Either sector would also be held responsible by governments should their modified organisms escape into the environment and proliferate.

Example: Proliferation of glyphosate resistance

File:Spread of Glyphosate Resistance.jpg
Since the introduction of glyphosate resistance in 1996, the amount of wild weeds resistant to glyphosate has exploded[5].

Since Monsanto's introduction of glyphosate resistance in 1996, there has been a growing concern over the affects of its widespread use. Glyphosate resistance is used primarily in Monsanto's Roundup Ready™ plants to work in tandem with Monsanto's Roundup™ herbicide, whose key active ingredient is glyphosate. Roundup has been used commercially since 1976. However, Roundup's true success did not truly begin until the development of Roundup Ready selective genetic markers.

It was at this time that an explosion in glyphosate resistant weeds began in the environment. Originally, there were no plants natively resistant to glyphosate. Even in 1996, 30 years after the introduction of Roundup, only 2 weeds worldwide had developed glyphosate resistance. However, as shown in the figure to the right, this number increased to 19 weeds by 2010[5]. The correlation between the introduction of commercial glyphosate resistance and the mutation of invasive weeds is substantial, and raised public concerns.

In 2006, after hearing a case brought against Monsanto by a coalition of farmers and environmental groups, the US District Court of Northern California ruled against Monsanto and issued an injunction on all Roundup Ready alfalfa sales. An appeal to the Ninth Circuit Court of Appeals upheld the District Court's ruling. It was not until a Supreme Court hearing in January of 2010 that the decision was reversed and the injunction lifted.

References

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  1. Error fetching PMID 21431833: [Giordano-Santini2011]
    Review article about selectable genetic markers as used in nematodes. Relatively new field for nematodes, possible due to the completion of the Caenorhabditis elegans genome.
  2. Error fetching PMID 20511419: [Poggi2010]
    Multiple antibiotic resistances as selectable genetic markers in Leptospira.
  3. Error fetching PMID 12697213: [Reinthaler2002]
    Development of antibiotic resistance in wild-type E. coli.
  4. Error fetching PMID 16916934: [Funke2006]
    Molecular basis of glyphosate resistance.
  5. Owen, M. [Owen2010]
    Report by Professor Micheal Owen from Iowa State University on glyphosate resistance for the U.S. House of representatives
  6. Zhang WJ, Yang SS, Shen XY, Jin YS, Zhao HJ and Wang T [Zhang2008]
    Salt tolerance as a selectable genetic marker.
  7. D. Parsons, M. Tolmasky, P. Chain and B. W. Segelke [Parsons2011]
    Report by the Lawrence Livermore National Laboratory on a new system for selectable markers
  8. Error fetching PMID 22251838: [Moen2011]
    Observes tumor growth in mice by introducing GFP into the mice.
  9. Error fetching PMID 8303295: [Chalfie1994]
    The first instance of using GFP as a marker.
  10. Error fetching PMID 21708017: [Ramsden2011]
    Use of inteins with GFP to introduce selectable markers into proteins.
  11. Twyman RM, Stroger E, Kohli A, Capell T and Christou P [Twyman2002]
    Various genetic markers used for artificial selection of rice crops.
  12. Error fetching PMID 15310922: [Goodwin2005]
    Herbicide selectable genetic markers in wheat.
All Medline abstracts: PubMed | HubMed
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