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[[Category:CH391L_S13]]
  
Ashley Kessel's first assignment. Cloned wiki.
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Ashley Kessel's first assignment: Cloned wiki (Feedback in discussion)
  
==Counterselectable Genetic Markers==
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==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.<cite>Cohen72</cite>
  
===Introduction===
+
===Screening vs. Selection===
In contrast to selection markers, counter-selection markers serve to eliminate unwanted elementsThese markers are often toxic or otherwise inhibitory to replication under certain conditionsSelective 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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The process of screening requires checking every cell (or colony in the case of bacteria) for the gene(s) of interestThis 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 markerScreening 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 genesSelectable 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.
===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 siteUpon 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 homologyThis event is often selected for by the presence of a selectable marker present on the vectorFollowing 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 surviveSince this recombination event is usually rare the counterselection step is essential to find the desired mutants.
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==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 [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 deathAlthough 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. coliAll 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-lactamasesDue 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>  
  
===Application: Plasmid curing===
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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]]]
  
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 replicationThese engineered origins permit replication at a permissive temperature but prevent replication upon switch to the counterselective temperatureAfter a period of growth at the selective temperature, the plasmid will be lost because it fails to replicate when the cell dividesAnother 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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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-lactamaseOne 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>
 
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=='''Parts'''==
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====tetAR====
 
  
The tetAR genes endow the cell with resistance to the antibiotic tetracycline by altering the cell membrane making it impermeable to the drugHowever, this alteration makes the cell hypersensitive to lipophilic chelating agents such as fusaric or quinalic acidsThis enables selection of cells that have lost the tetAR genes by exposure to fusaric acid<cite>Bochner1980</cite>.
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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 howeverAdditionally, multiple antibiotics may also be necessary to simultaneously select for markers on separate plasmidsFor more information about how they work, see the links below: <br>
 +
[http://en.wikipedia.org/wiki/Kanamycin Kanamycin on Wikipedia] <br>
 +
[http://en.wikipedia.org/wiki/Chloramphenicol Chloramphenicol on Wikipedia] <br>
  
====sacB====
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==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 [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> 
  
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 structure of tetracycline. [http://en.wikipedia.org/wiki/Tetracycline Tetracycline on Wikipedia]]]
  
====rpsL====
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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>
  
The rpsL gene encodes the S12 protein component of the 30S ribosomeCertain mutations of the gene cause resistance to the antibiotic streptomycin which targets the 30S ribosomeResistance 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 alleleSelection on streptomycin will only permit those cells that have lost the wild-type gene to survive.
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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 saltsAlthough ''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 efficiencyCheap, non-toxic Nickel Chloride is the most commonly used selection agentAlthough 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>
  
====ccdB====
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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>.
  
[[Image:Gateway_cloning.gif‎ | Invitrogen "Gateway" cloning system| right |thumb|200px]]
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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 transporterThis 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>.
ccdB is the toxin component of the toxin-antitoxin system of the F plasmidccdB 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 strainThis 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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[[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]]]
  
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 UracilSupplying 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 markerURA3 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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===Using a ''tetA'' Fusion Protein for Monitoring Selection===
 +
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 productTo 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 mediaGFP 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 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'').
  
It is important to note that the URA3/5-FOA system only works in ''Saccharomyces cerevisiae'' in strains with a chromosomal URA3 mutationRecently 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.
+
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 mediaRecent 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>.
  
====pheS====
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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]]
  
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 eliminatedUnlike 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 presentThis strategy has been used in both Gram-negative and Gram-positive bacteria.
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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 cellsGrowth 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 strainThus, selections with GFAT markers are limited to minimal media, but the addition of tryptone increases growth rates while still allowing selectionUnder these conditions, selection for ''glmS'' was shown to be an effective replacement for ''ampR'' selection<cite>Wu11</cite>.
  
====Thymidine Kinase====
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==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.
  
Thymidine kinase (TK) is a popular counterselection tool used in mammalian cell cultureTK 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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Another area of ongoing progress is the discovery of non-antibiotic markers to potentially replace commonly used genes like ''ampR'' in bacterial engineeringAlthough 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 alarmIf 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.
  
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.
+
See Also: <br>
 +
[http://openwetware.org/wiki/CH391L/S12/Selectablegeneticmarkers Selectable Genetic Markers] <br>
 +
[http://openwetware.org/wiki/CH391L/S12/ToxinAntitoxins Toxin/Antitoxin Systems] <br>
 +
[http://openwetware.org/wiki/CH391L/S12/CounterSelection Counterselection] <br>
  
Cells containing the Thymidine kinase gene can then be counterselected against by the addition of toxic Thymidine analogs such as 5-bromo-deoxyuridine or GanciclovirThese Thymidine analogs are chain terminators which do not have a hydroxyl group in the 3'-position which is required for continued chain elongationThus, cells with TK will be eliminated due to their inability to synthesize complete DNA sequences.
+
==iGEM Connections==
 +
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 ribozymePortions 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].
  
  
=='''Nutritional'''==
+
== References ==  
  
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''.
 
 
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].
 
 
==References==
 
 
<biblio>
 
<biblio>
#Bochner1980 pmid=6259126
+
#Cohen72 pmid=4559594
#Gay1985 pmid=2997137
+
//CaCl<sub>2</sub> bacterial transformations
#Lederberg1951 pmid=14832197
+
#Collins06 pmid=16715074
#Bernard1992 pmid=1324324
+
//Dual selection with separate markers
#Kast1992 pmid=8125286
+
#McNicholas92 pmid=1459940
#Boeke1984 pmid=6394957
+
//The tetA(C) gene from pBR322
#Galvao2005 pmid=15691944
+
#Muranaka09 pmid=19190095
 +
//Riboswitch selection/screening using a tetA-GFP fusion marker
 +
#Nomura07 pmid=17944473
 +
//Reengineering the TPP riboswitch using ''tetA'' dual selection
 +
#Podolsky96 pmid=8954882
 +
//Nickel selection with ''tetA''
 +
#Sutcliffe78 pmid=358200
 +
//Background on the ''ampR'' gene from pBR322
 +
#Wu11 pmid=21340036
 +
//GFAT as a selectable marker
 +
</biblio>

Latest revision as of 20:26, 28 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

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