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

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Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)
 
Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)
  
==Selectable Markers Overview==
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==Introduction==
[[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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The ability to introduce exogenous DNA into an organism to alter its genetic program is one of the most crucial tools in modern biology. Early work showed that certain bacteria could acquire the traits of a related strain through the addition of heat-killed cells.  Although it was not well understood at the time, the transfer of gene-encoding DNA from one strain to another facilitated this.  This concept was turned into a useful tool upon the advent of bacterial plasmid transformations in the early 1970's, which allowed genes of interest to be easily inserted into ''E. coli''.  Over the years, methods have been developed to introduce exogenous genes into a wide range of useful organisms, including bacteria, yeasts, plants, and animal tissues.  These methods vary enormously in efficiency however, necessitating a way to identify and isolate cells which contain the DNA of interest.  This can be accomplished either by screening for successfully modified cells, or through selection.<cite>Cohen72</cite>
  
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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===Screening vs. Selection===
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The process of screening requires checking every cell (or colony in the case of bacteria) for the gene(s) of interest. This is often done with the aid of a screenable marker, like a fluorescent protein expressing gene, that allows rapid identification of transformed cells. Alternatively, exogenous genes in bacterial colonies can be detected by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] or a variety of other methods without the use of a marker.  Screening is often time-consuming and expensive, particularly when the fraction of modified cells is low.  A much more efficient strategy, selection, involves growing cells in conditions which only allow for survival of those with the desired genes.  Selectable marker genes can be combined with any other genes of interest and used to select for their insertion without additional screening steps.  In bacteria, the most commonly used selectable markers provide resistance to antibiotics, allowing for positive selection of plasmids following transformation.
  
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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==Antibiotic Resistance Markers: ''ampR''==
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Perhaps the most commonly used selectable marker in bacteria is the ''ampR'' gene, which provides resistance to certain beta-lactam antibiotics such as [http://en.wikipedia.org/wiki/Ampicillin ampicillin] (amp) and its more stable relative [http://en.wikipedia.org/wiki/Carbenicillin carbenicillin] (carb).  [http://en.wikipedia.org/wiki/Beta-lactam_antibiotic Beta-lactam antibiotics] are [http://en.wikipedia.org/wiki/Penicillin penicillin] derivatives that inhibit synthesis of bacterial [http://en.wikipedia.org/wiki/Peptidoglycan peptidoglycan] cell walls, arresting cell division and ultimately leading to cell death.  Although beta-lactams are primarily functional against gram-positive bacteria due to their larger cell walls, some examples, such as amp and carb, are capable of killing gram-negative E. coli. All antibiotics in the family share a central four atom ring structure known as the beta-lactam ring, which serves as a cleavage target for enzymes known as beta-lactamases. Due to the shared structural features of beta-lactam antibiotics, these enzymes often have promiscuous activities that target multiple drugs.  Upon cleavage of the lactam ring, antibiotics such as amp lose their toxicity, allowing cell growth and division to resume.<cite>Sutcliffe78</cite>
  
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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[[Image:amp.png|thumb|center|500px| The structure of ampicillin, including the central beta-lactam ring cleaved by the ''ampR'' gene product [http://en.wikipedia.org/wiki/Ampicillin Ampicillin on Wikipedia]]]
  
==Types of Selectable Markers==
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The common ''ampR'' gene, as used in the ''E. coli'' pBR322 plasmid, was naturally derived from [http://en.wikipedia.org/wiki/Salmonella ''Salmonella''] bacteria through [http://en.wikipedia.org/w/index.php?title=Transposition_(horizontal_gene_transfer)&redirect=no transposition].  Because it efficiently cleaves amp and carb, it's one of the most useful markers in ''E. coli''.  Ampicillin does not kill the bacteria quickly, so cells transformed with the ''ampR'' marker can be immediately plated on selective media, before they've produced a significant amount of beta-lactamase.  One downside to the use of amp is that it's rapid removal from selective media by beta-lactamase can allow for growth of cells that lack resistance.  This is often witnessed in the form of satellite colonies on amp plates.  The use of carbenicillin for selection reduces the problem, as carb is less rapidly degraded by the ''ampR'' beta-lactamase, but carb is significantly more expensive than amp. <cite>Sutcliffe78</cite> <br>
  
===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.
 
  
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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Marker genes that provide resistance to [http://en.wikipedia.org/wiki/Kanamycin kanamycin] (kan) and [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol] (cap) are also popular for use in ''E. coli'', as kan and cap are more stable than ampicillin. Bacterial cells transformed with kan and cap resistance markers must be allowed to recover before plating however.  Additionally, multiple antibiotics may also be necessary to simultaneously select for markers on separate plasmids.  For more information about how they work, see the links below: <br>
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[http://en.wikipedia.org/wiki/Kanamycin Kanamycin on Wikipedia] <br>
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[http://en.wikipedia.org/wiki/Chloramphenicol Chloramphenicol on Wikipedia] <br>
  
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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==Antibiotic/Non-Antibiotic Dual Selection: ''tetA(C)''==
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===Tetracycline Positive Selection===
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The ''tetA(C)'' gene is primarily used for positive selection in bacteria, similar to ''ampR''. ''tetA(C)'' encodes a membrane-bound transporter that rapidly pumps the antibiotic [http://en.wikipedia.org/wiki/Tetracycline tetracycline] out of bacterial cells. This process is energy dependent, as the protein uses the influx of H+ ions from the surrounding environment to drive the process.  Tetracycline is a broad-spectrum, polyketide antibiotic derived from ''Streptomyces'' that inhibits bacterial translation. The antibiotic binds the 30s subunit of bacterial ribosomes, blocking entry of aminoacyl-tRNAs to the A site of the ribosome.<cite>McNicholas92</cite>
  
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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[[Image:tet.png|thumb|center|500px| The structure of tetracycline. [http://en.wikipedia.org/wiki/Tetracycline Tetracycline on Wikipedia]]]
  
===Herbicidal===
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Because tetracycline resistance is gained through expression of a transporter and not a modifying enzyme, selective media maintains its antibiotic levels during growth of resistant cells.  High levels of ''tetA'' expression can have a detrimental effect on the cells however, due the energy cost of pumping tetracycline and increased vulnerability to certain extracellular conditions.  This often results in fewer viable cells when using tetracycline selection compared to selection with antibiotics like ampicillin and kanamycin.<cite>Podolsky96</cite>
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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===Nickel Salt Negative Selection===
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Expression of ''tetA'' in bacteria has the side effect of making cells more vulnerable to poisoning by either lipophilic chelating agents or metal salts.  Although ''tetA'' expressing ''Salmonella'' strains were shown to be extremely sensitive to chelating agents such as fusaric or quinaldic acids, these compounds are only marginally effective on ''E. coli''.  Based on earlier observations that metals like cadmium inhibited growth of tetracycline-resistant bacteria, a technique was developed that uses nickel salts to select against ''tetA'' expressing E. coli with much greater efficiency. Cheap, non-toxic Nickel Chloride is the most commonly used selection agent.  Although originally used to select for cells that had lost a ''tetA'' marker, this method has proven useful in dual selection schemes for evolving regulators of gene expression. <cite>Podolsky96,Nomura07</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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===Engineering Riboswitches using ''tetA'' Dual Selection===
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The evolution of functional [http://www.openwetware.org/wiki/CH391L/S13/Riboswitches Riboswitches] requires selection of a library of mutants in both an ON state and an OFF state. This removes variants that either activate or repress gene expression regardless of the small molecule used to regulate them. Previous attempts at dual selection required the use of both positive and negative selection markers. This necessitated intermediate steps to purify plasmids from the pool and re-transform them along with either the positive or negative marker.  Aside from being labor intensive, this method also increased the rate of false positives in the pool <cite>Collins06,Muranaka09</cite>.
  
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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The fact that a single ''tetA'' gene can be used for both positive and negative selection greatly simplifies the process of selecting for riboswitches, as a culture of cells expressing a library of riboswitches can be alternately swapped between media that selects for or against expression of the tetracycline transporter.  This strategy was used by the [http://yokobayashilab.net/ Yokobayashi Lab] at [http://www.ucdavis.edu UC Davis] to select for mutants of the ''E. coli'' TPP riboswitch (from ''thiM'') that activated downstream gene expression in the presence of thiamine instead of repressing it. A library of TPP riboswitch variants was cloned upstream of a ''tetA'' marker and transformed into ''E. coli''.  The first round of selection involved plating transformants on media with thiamine and tetracycline to select for mutants that were turned ON.  The survivors were then transferred to media with nickel chloride and no thiamine to remove any variants that did not turn off in the absence of thiamine.  A similar approach can be carried out in liquid culture, as shown below.<cite>Nomura07</cite>.
  
===Auxotrophic===
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[[Image:DualSelection.png|thumb|center|550px| A ''tetA'' dual selection scheme as used by the [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit4 2011 Peking R iGEM team] to select for ribozymes based on a TPP aptamer fused to the hammerhead ribozyme.  A similar scheme was used to switch the ON/OFF behavior of the TPP riboswitch. [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit4 Team Website]]]
  
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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===Using a ''tetA'' Fusion Protein for Monitoring Selection===
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More recent work by the [http://yokobayashilab.net/ Yokobayashi Lab] involved combining the ''tetA'' marker with GFP to allow screening of ''thiM'' riboswitch function following each round of dual selection. The ''tetA'' transporter remains functional when GFP is fused to its c-terminal end, facilitating selection and screening steps with the same gene product. To measure function of different riboswitch variants in the selection pool, samples were plated on LB and individual colonies were picked and used to seed culture in non-selective media. GFP fluorescence of each culture was measured to determine the amount of gene expression generated by that particular riboswitch, and functional variants were sequenced<cite>Muranaka09</cite>.  
  
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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==Auxotrophic Marker Selection: GFAT==
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An alternative method of positive selection involves the use of auxotrophs, or cells that lack the ability to synthesize a compound necessary for their own growth.  In bacteria and yeast, useful auxotrophic strains are easily generated by knocking out a single gene involved in a chemical synthesis pathway.  The resulting strains can be grown in media containing the essential product but require complementation with an exogenous copy of the missing gene (the auxotrophic marker) to remain viable in media lacking it.  Auxotrophic markers are extremely useful in yeast, where the range of useful antibiotic markers is limited, but they are also used in bacterial applications where introduction of antibiotic-resistance genes could be problematic. Recently, a single gene involved in glucosamine synthesis was shown to be a useful auxotrophic marker in ''E. coli'' and fission yeast (''S. pombe'').
  
===Other===
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Glutamine:fructose-6-phosphate aminotransferase (GFAT) is an enzyme required for the biosynthesis of hexosamines in both bacteria and eukaryotes. GFAT catalyzes the rate-limiting first step in the pathway, converting fructose-6-phosphate into glucosamine-6-phosphate.  In ''E. coli'', GFAT is expressed from the ''glmS'' gene, which is necessary for growth in minimal media.  Recent work has shown that ''E. coli'' lacking ''glmS'' are capable of growing at near wild type levels when supplemented with glucosamine however, and complementation with a plasmid-encoded ''glmS'' gene restores hexosamine synthesis and growth<cite>Wu11</cite>.
[[Image:Alternative Selective Marker.jpg|thumb|left|An alternative technique for selectable markers that avoids antibiotic resistance<cite>Parsons2011</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.
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[[Image:GFAT.png|thumb|center|400px| '''The growth condition of E. coli K12 ΔglmS in different medium.''' (A) The growth condition of E. coli K12 ΔglmS in M9 minimal medium supplemented with glucosamine and different concentration of yeast extract respectively. (B) The growth condition of E. coli K12 ΔglmS in MmGTV, MT, MG, MGTV, MGT medium (M: M9 medium, Mm: M9 medium subtracting Mg2+ and glucose, G: glucosamine, T: tryptone, V: vitamin mixture)<cite>Wu11</cite>. doi:10.1371/journal.pone.0017082.g004]]
  
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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Selection of ''glmS'' expressing cells is complicated by the fact that rich media such as LB contains sufficient glucosamine to support limited growth of ''glmS'' knockout cells. Growth tests using M9 minimal media with individual LB components added to it showed that yeast extract is the source of glucosamine that allows growth of the knockout strain. Thus, selections with GFAT markers are limited to minimal media, but the addition of tryptone increases growth rates while still allowing selection. Under these conditions, selection for ''glmS'' was shown to be an effective replacement for ''ampR'' selection<cite>Wu11</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.
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==Future Directions==
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Although the currently available set of selectable markers has helped us to overcome many of the challenges involved in synthetic biology, the continued discovery of new markers may greatly simplify the process of designing organisms with useful functions. The variety of useful markers available for engineering of higher eukaryotes, including animals and human tissue culture lines pales in comparison to those available for use in bacteria or even yeast.  Thus, the discovery of new selection schemes may allow experiments that are not currently feasible.
  
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Another area of ongoing progress is the discovery of non-antibiotic markers to potentially replace commonly used genes like ''ampR'' in bacterial engineering.  Although antibiotic resistance is an increasingly common trait in wild bacteria, the possibility of engineered organisms transferring their resistance to otherwise susceptible bacteria is a frequent cause for alarm.  If engineered bacteria are to prove useful for tasks like bio-remediation, we will have to engineer them without the benefit of antibiotic selection or markers that require growth in minimal media.
  
==Types of Screening==
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See Also: <br>
[[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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[http://openwetware.org/wiki/CH391L/S12/Selectablegeneticmarkers Selectable Genetic Markers] <br>
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[http://openwetware.org/wiki/CH391L/S12/ToxinAntitoxins Toxin/Antitoxin Systems] <br>
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[http://openwetware.org/wiki/CH391L/S12/CounterSelection Counterselection] <br>
  
===Blue/White Screening===
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==iGEM Connections==
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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The [http://2011.igem.org/Team:Peking_R 2011 Peking_R iGEM team] from Peking University used ''tetA'' dual selection to engineer a ribozyme-based "genetic rheostat."  Their approach involved fusing the TPP aptamer domain from the ''thiM'' riboswitch to a section of the hammerhead ribozyme.  Portions of the construct were randomized and selections were carried out in a similar fashion to those performed by the [http://yokobayashilab.net/ Yokobayashi Lab]<cite>Muranaka09</cite>.  This approach did increase the ratio of ON/OFF ribozymes in their pool, and similar selections using an adenine aptamer domain also demonstrated the potential of the [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit4 approach].
  
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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== References ==
  
===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>.]]
 
 
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.
 
 
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>.
 
 
 
==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>.
 
 
 
==Issues==
 
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.
 
 
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==
 
 
<biblio>
 
<biblio>
#Giordano-Santini2011 pmid=21431833
+
#Cohen72 pmid=4559594
//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.
+
//CaCl<sub>2</sub> bacterial transformations
 
+
#Collins06 pmid=16715074
#Moen2011 pmid=22251838
+
//Dual selection with separate markers
//Observes tumor growth in mice by introducing GFP into the mice.
+
#McNicholas92 pmid=1459940
 
+
//The tetA(C) gene from pBR322
#Chalfie1994 pmid=8303295
+
#Muranaka09 pmid=19190095
//The first instance of using GFP as a marker.
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//Riboswitch selection/screening using a tetA-GFP fusion marker
 
+
#Nomura07 pmid=17944473
#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]
+
//Reengineering the TPP riboswitch using ''tetA'' dual selection
//Various genetic markers used for artificial selection of rice crops.
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#Podolsky96 pmid=8954882
 
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//Nickel selection with ''tetA''
#Parsons2011 [https://e-reports-ext.llnl.gov/pdf/476269.pdf D. Parsons, M. Tolmasky, P. Chain and B. W. Segelke]
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#Sutcliffe78 pmid=358200
//Report by the Lawrence Livermore National Laboratory on a new system for selectable markers
+
//Background on the ''ampR'' gene from pBR322
 
+
#Wu11 pmid=21340036
#Poggi2010 pmid=20511419
+
//GFAT as a selectable marker
//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>
 
</biblio>

Revision as of 21:59, 24 January 2014


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

Contents

Introduction

The ability to introduce exogenous DNA into an organism to alter its genetic program is one of the most crucial tools in modern biology. Early work showed that certain bacteria could acquire the traits of a related strain through the addition of heat-killed cells. Although it was not well understood at the time, the transfer of gene-encoding DNA from one strain to another facilitated this. This concept was turned into a useful tool upon the advent of bacterial plasmid transformations in the early 1970's, which allowed genes of interest to be easily inserted into E. coli. Over the years, methods have been developed to introduce exogenous genes into a wide range of useful organisms, including bacteria, yeasts, plants, and animal tissues. These methods vary enormously in efficiency however, necessitating a way to identify and isolate cells which contain the DNA of interest. This can be accomplished either by screening for successfully modified cells, or through selection.[1]

Screening vs. Selection

The process of screening requires checking every cell (or colony in the case of bacteria) for the gene(s) of interest. This is often done with the aid of a screenable marker, like a fluorescent protein expressing gene, that allows rapid identification of transformed cells. Alternatively, exogenous genes in bacterial colonies can be detected by PCR or a variety of other methods without the use of a marker. Screening is often time-consuming and expensive, particularly when the fraction of modified cells is low. A much more efficient strategy, selection, involves growing cells in conditions which only allow for survival of those with the desired genes. Selectable marker genes can be combined with any other genes of interest and used to select for their insertion without additional screening steps. In bacteria, the most commonly used selectable markers provide resistance to antibiotics, allowing for positive selection of plasmids following transformation.

Antibiotic Resistance Markers: ampR

Perhaps the most commonly used selectable marker in bacteria is the ampR gene, which provides resistance to certain beta-lactam antibiotics such as ampicillin (amp) and its more stable relative carbenicillin (carb). Beta-lactam antibiotics are penicillin derivatives that inhibit synthesis of bacterial peptidoglycan cell walls, arresting cell division and ultimately leading to cell death. Although beta-lactams are primarily functional against gram-positive bacteria due to their larger cell walls, some examples, such as amp and carb, are capable of killing gram-negative E. coli. All antibiotics in the family share a central four atom ring structure known as the beta-lactam ring, which serves as a cleavage target for enzymes known as beta-lactamases. Due to the shared structural features of beta-lactam antibiotics, these enzymes often have promiscuous activities that target multiple drugs. Upon cleavage of the lactam ring, antibiotics such as amp lose their toxicity, allowing cell growth and division to resume.[2]

File:Amp.png
The structure of ampicillin, including the central beta-lactam ring cleaved by the ampR gene product Ampicillin on Wikipedia

The common ampR gene, as used in the E. coli pBR322 plasmid, was naturally derived from Salmonella bacteria through transposition. Because it efficiently cleaves amp and carb, it's one of the most useful markers in E. coli. Ampicillin does not kill the bacteria quickly, so cells transformed with the ampR marker can be immediately plated on selective media, before they've produced a significant amount of beta-lactamase. One downside to the use of amp is that it's rapid removal from selective media by beta-lactamase can allow for growth of cells that lack resistance. This is often witnessed in the form of satellite colonies on amp plates. The use of carbenicillin for selection reduces the problem, as carb is less rapidly degraded by the ampR beta-lactamase, but carb is significantly more expensive than amp. [2]


Marker genes that provide resistance to kanamycin (kan) and chloramphenicol (cap) are also popular for use in E. coli, as kan and cap are more stable than ampicillin. Bacterial cells transformed with kan and cap resistance markers must be allowed to recover before plating however. Additionally, multiple antibiotics may also be necessary to simultaneously select for markers on separate plasmids. For more information about how they work, see the links below:
Kanamycin on Wikipedia
Chloramphenicol on Wikipedia

Antibiotic/Non-Antibiotic Dual Selection: tetA(C)

Tetracycline Positive Selection

The tetA(C) gene is primarily used for positive selection in bacteria, similar to ampR. tetA(C) encodes a membrane-bound transporter that rapidly pumps the antibiotic tetracycline out of bacterial cells. This process is energy dependent, as the protein uses the influx of H+ ions from the surrounding environment to drive the process. Tetracycline is a broad-spectrum, polyketide antibiotic derived from Streptomyces that inhibits bacterial translation. The antibiotic binds the 30s subunit of bacterial ribosomes, blocking entry of aminoacyl-tRNAs to the A site of the ribosome.[3]

The structure of tetracycline. Tetracycline on Wikipedia

Because tetracycline resistance is gained through expression of a transporter and not a modifying enzyme, selective media maintains its antibiotic levels during growth of resistant cells. High levels of tetA expression can have a detrimental effect on the cells however, due the energy cost of pumping tetracycline and increased vulnerability to certain extracellular conditions. This often results in fewer viable cells when using tetracycline selection compared to selection with antibiotics like ampicillin and kanamycin.[4]

Nickel Salt Negative Selection

Expression of tetA in bacteria has the side effect of making cells more vulnerable to poisoning by either lipophilic chelating agents or metal salts. Although tetA expressing Salmonella strains were shown to be extremely sensitive to chelating agents such as fusaric or quinaldic acids, these compounds are only marginally effective on E. coli. Based on earlier observations that metals like cadmium inhibited growth of tetracycline-resistant bacteria, a technique was developed that uses nickel salts to select against tetA expressing E. coli with much greater efficiency. Cheap, non-toxic Nickel Chloride is the most commonly used selection agent. Although originally used to select for cells that had lost a tetA marker, this method has proven useful in dual selection schemes for evolving regulators of gene expression. [4, 5]

Engineering Riboswitches using tetA Dual Selection

The evolution of functional Riboswitches requires selection of a library of mutants in both an ON state and an OFF state. This removes variants that either activate or repress gene expression regardless of the small molecule used to regulate them. Previous attempts at dual selection required the use of both positive and negative selection markers. This necessitated intermediate steps to purify plasmids from the pool and re-transform them along with either the positive or negative marker. Aside from being labor intensive, this method also increased the rate of false positives in the pool [6, 7].

The fact that a single tetA gene can be used for both positive and negative selection greatly simplifies the process of selecting for riboswitches, as a culture of cells expressing a library of riboswitches can be alternately swapped between media that selects for or against expression of the tetracycline transporter. This strategy was used by the Yokobayashi Lab at UC Davis to select for mutants of the E. coli TPP riboswitch (from thiM) that activated downstream gene expression in the presence of thiamine instead of repressing it. A library of TPP riboswitch variants was cloned upstream of a tetA marker and transformed into E. coli. The first round of selection involved plating transformants on media with thiamine and tetracycline to select for mutants that were turned ON. The survivors were then transferred to media with nickel chloride and no thiamine to remove any variants that did not turn off in the absence of thiamine. A similar approach can be carried out in liquid culture, as shown below.[5].

File:DualSelection.png
A tetA dual selection scheme as used by the 2011 Peking R iGEM team to select for ribozymes based on a TPP aptamer fused to the hammerhead ribozyme. A similar scheme was used to switch the ON/OFF behavior of the TPP riboswitch. Team Website

Using a tetA Fusion Protein for Monitoring Selection

More recent work by the Yokobayashi Lab involved combining the tetA marker with GFP to allow screening of thiM riboswitch function following each round of dual selection. The tetA transporter remains functional when GFP is fused to its c-terminal end, facilitating selection and screening steps with the same gene product. To measure function of different riboswitch variants in the selection pool, samples were plated on LB and individual colonies were picked and used to seed culture in non-selective media. GFP fluorescence of each culture was measured to determine the amount of gene expression generated by that particular riboswitch, and functional variants were sequenced[7].

Auxotrophic Marker Selection: GFAT

An alternative method of positive selection involves the use of auxotrophs, or cells that lack the ability to synthesize a compound necessary for their own growth. In bacteria and yeast, useful auxotrophic strains are easily generated by knocking out a single gene involved in a chemical synthesis pathway. The resulting strains can be grown in media containing the essential product but require complementation with an exogenous copy of the missing gene (the auxotrophic marker) to remain viable in media lacking it. Auxotrophic markers are extremely useful in yeast, where the range of useful antibiotic markers is limited, but they are also used in bacterial applications where introduction of antibiotic-resistance genes could be problematic. Recently, a single gene involved in glucosamine synthesis was shown to be a useful auxotrophic marker in E. coli and fission yeast (S. pombe).

Glutamine:fructose-6-phosphate aminotransferase (GFAT) is an enzyme required for the biosynthesis of hexosamines in both bacteria and eukaryotes. GFAT catalyzes the rate-limiting first step in the pathway, converting fructose-6-phosphate into glucosamine-6-phosphate. In E. coli, GFAT is expressed from the glmS gene, which is necessary for growth in minimal media. Recent work has shown that E. coli lacking glmS are capable of growing at near wild type levels when supplemented with glucosamine however, and complementation with a plasmid-encoded glmS gene restores hexosamine synthesis and growth[8].

File:GFAT.png
The growth condition of E. coli K12 ΔglmS in different medium. (A) The growth condition of E. coli K12 ΔglmS in M9 minimal medium supplemented with glucosamine and different concentration of yeast extract respectively. (B) The growth condition of E. coli K12 ΔglmS in MmGTV, MT, MG, MGTV, MGT medium (M: M9 medium, Mm: M9 medium subtracting Mg2+ and glucose, G: glucosamine, T: tryptone, V: vitamin mixture)[8]. doi:10.1371/journal.pone.0017082.g004

Selection of glmS expressing cells is complicated by the fact that rich media such as LB contains sufficient glucosamine to support limited growth of glmS knockout cells. Growth tests using M9 minimal media with individual LB components added to it showed that yeast extract is the source of glucosamine that allows growth of the knockout strain. Thus, selections with GFAT markers are limited to minimal media, but the addition of tryptone increases growth rates while still allowing selection. Under these conditions, selection for glmS was shown to be an effective replacement for ampR selection[8].

Future Directions

Although the currently available set of selectable markers has helped us to overcome many of the challenges involved in synthetic biology, the continued discovery of new markers may greatly simplify the process of designing organisms with useful functions. The variety of useful markers available for engineering of higher eukaryotes, including animals and human tissue culture lines pales in comparison to those available for use in bacteria or even yeast. Thus, the discovery of new selection schemes may allow experiments that are not currently feasible.

Another area of ongoing progress is the discovery of non-antibiotic markers to potentially replace commonly used genes like ampR in bacterial engineering. Although antibiotic resistance is an increasingly common trait in wild bacteria, the possibility of engineered organisms transferring their resistance to otherwise susceptible bacteria is a frequent cause for alarm. If engineered bacteria are to prove useful for tasks like bio-remediation, we will have to engineer them without the benefit of antibiotic selection or markers that require growth in minimal media.

See Also:
Selectable Genetic Markers
Toxin/Antitoxin Systems
Counterselection

iGEM Connections

The 2011 Peking_R iGEM team from Peking University used tetA dual selection to engineer a ribozyme-based "genetic rheostat." Their approach involved fusing the TPP aptamer domain from the thiM riboswitch to a section of the hammerhead ribozyme. Portions of the construct were randomized and selections were carried out in a similar fashion to those performed by the Yokobayashi Lab[7]. This approach did increase the ratio of ON/OFF ribozymes in their pool, and similar selections using an adenine aptamer domain also demonstrated the potential of the approach.


References

Error fetching PMID 4559594:
Error fetching PMID 16715074:
Error fetching PMID 1459940:
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Error fetching PMID 8954882:
Error fetching PMID 358200:
Error fetching PMID 21340036:
  1. Error fetching PMID 4559594: [Cohen72]
    CaCl2 bacterial transformations
  2. Error fetching PMID 358200: [Sutcliffe78]
    Background on the ampR gene from pBR322
  3. Error fetching PMID 1459940: [McNicholas92]
    The tetA(C) gene from pBR322
  4. Error fetching PMID 8954882: [Podolsky96]
    Nickel selection with tetA
  5. Error fetching PMID 17944473: [Nomura07]
    Reengineering the TPP riboswitch using tetA dual selection
  6. Error fetching PMID 16715074: [Collins06]
    Dual selection with separate markers
  7. Error fetching PMID 19190095: [Muranaka09]
    Riboswitch selection/screening using a tetA-GFP fusion marker
  8. Error fetching PMID 21340036: [Wu11]
    GFAT as a selectable marker
All Medline abstracts: PubMed | HubMed