CH391L/S12/CounterSelection: Difference between revisions

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====URA3====
====URA3====


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.  Many common yeast strains contain a mutation in the URA3 gene making the cells auxotrophic for Uracil.  Supplying an intact copy of the URA3 gene on a plasmid or integration cassette restores prototrophy and can be used to select for cells which have taken up the vector containing the marker.  URA3 is also known to convert 5-Fluoroorotic acid (5-FOA) into the toxic compound 5-fluorouracil leading to cell death.  Therefore, counterselection on 5-FOA will select for clones which have lost the URA3 marker.
The URA3 gene from ''Saccharomyces cerevisiae'' encodes for orotidine-5'-monophosphate decarboxylase which is involved in de novo synthesis of pyrimidine nucleotides (pathway [http://pathway.yeastgenome.org/YEAST/NEW-IMAGE?type=PATHWAY&object=PYRIMID-RNTSYN-PWY&detail-level=2]).  URA3 normally catalyzes the decarboxylation of orotidine 5-phosphate (OMP) to uridylic acid (UMP).  The gene is especially useful as a marker in that it can be used for both selection and counterselection<cite>Boeke1984</cite>.  Many common yeast strains contain a mutation in the URA3 gene making the cells auxotrophic for Uracil.  Supplying an intact copy of the URA3 gene on a plasmid or integration cassette restores prototrophy and can be used to select for cells which have taken up the vector containing the marker.  URA3 is also known to convert 5-Fluoroorotic acid (5-FOA) into the toxic compound 5-fluorouracil leading to cell death.  Therefore, counterselection on 5-FOA will select for clones which have lost the URA3 marker.




It is important to note that the URA3/5-FOA system only works in ''Saccharomyces cerevisiae'' in strains with a chromosomal URA3 mutation.  Recently the system has been ported to bacterial species.  By identifying the URA3 homolog, (pyrF in ''E.coli'' and ''p.putida'') and deleting it, URA3 selection/counterseletion can be employed.
It is important to note that the URA3/5-FOA system only works in ''Saccharomyces cerevisiae'' in strains with a chromosomal URA3 mutation.  Recently the system has been ported to bacterial species<cite>Galvao2005</cite>.  By identifying the URA3 homolog, (pyrF in ''E.coli'' and ''p.putida'') and deleting it, URA3 selection/counterseletion can be employed.


====pheS====
====pheS====
Line 44: Line 44:
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.  When grown on media containing p-chlorophenylalanine cells harboring the mutant version of the gene will be eliminated.  Unlike the rpsL counterselection, the mutants pheS phenotype is dominant meaning that the presence of the mutant gene will still cause toxicity when the wild-type version is also present.  This strategy has been used in both Gram-negative and Gram-positive bacteria.
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.  When grown on media containing p-chlorophenylalanine cells harboring the mutant version of the gene will be eliminated.  Unlike the rpsL counterselection, the mutants pheS phenotype is dominant meaning that the presence of the mutant gene will still cause toxicity when the wild-type version is also present.  This strategy has been used in both Gram-negative and Gram-positive bacteria.


===References===
==References==
<biblio>
<biblio>
#Boeke1984 pmid=6394957
#Galvao2005 pmid=15691944

Revision as of 16:32, 18 February 2012


Counterselectable Genetic Markers

Introduction

In contrast to selection markers, counter-selection markers serve to eliminate unwanted elements. These markers are often toxic or otherwise inhibitory to replication under certain conditions. Selective conditions often involve exposure to a specific substrates or shift in growth conditions. These elements are often incorporated into genetic modification schemes in order to select for rare recombination events that require the removal of the marker or to selectively eliminate plasmids or cells from a given population.

Application: Allelic Exchange

Allelic Exchange. Marx C.J. BMC Research Notes. 2008

The introduction of specific mutations in a genetic sequence is a powerful way to learn about gene function or to engineer an organism for a desired application. A common way to introduce specific mutations into a target sequence is through allelic exchange. In a typical allelic exchange experiment, the chromosomal sequence to be mutated is synthesized and cloned onto a vector. This sequence is either highly homologous (except for the introduced mutations) to the chromosomal version or else is flanked by homologous sequences specifying the desired insertion site. Upon introduction to the cell, the cell’s homologous recombination machinery will recognize the sites of homology between the vector and the chromosome and at some frequency will stimulate the integration of the vector at the site of homology. This event is often selected for by the presence of a selectable marker present on the vector. Following this selection, it is often desirable to remove the vector to produce a “seemless” insertion. For this purpose, a counterselectable marker is included on the vector. Upon induction of the counterselectable condition, only those cells that have excised the vector sequence through a second recombination event will survive. Since this recombination event is usually rare the counterselection step is essential to find the desired mutants.

Application: Plasmid curing

It is often necessary to "cure" or isolate cells that have lost a plasmid. This can typically be achieved through serial passage in non-selective media and screening for plasmid loss. Counterselective markers can be incorporated onto plasmids so that their loss can be selected for. A common strategy includes the use of a temperature sensitive origin of replication. These engineered origins permit replication at a permissive temperature but prevent replication upon switch to the counterselective temperature. After a period of growth at the selective temperature, the plasmid will be lost because it fails to replicate when the cell divides. Another strategy involves the inclusion of a counterselectable marker (see "parts" below) on the vector allowing for selection of cells that lose the plasmid on their own.

Parts

tetAR

The tetAR genes endow the cell with resistance to the antibiotic tetracycline by altering the cell membrane making it impermeable to the drug. However, this alteration makes the cell hypersensitive to lipophilic chelating agents such as fusaric or quinalic acids. This enables selection of cells that have lost the tetAR genes by exposure to fusaric acid.

sacB

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. 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.

rpsL

The rpsL gene encodes the S12 protein component of the 30S ribosome. Certain mutations of the gene cause resistance to the antibiotic streptomycin which targets the 30S ribosome. Resistance to streptomycin resistance is recessive meaning that an additional wild-type copy of the gene will lead to streptomycin sensitivity. Therefore, the wild-type rpsL gene can be used as a counterselective marker in a strain that already possesses a mutant allele. Selection on streptomycin will only permit those cells that have lost the wild-type gene to survive.

ccdB

Invitrogen "Gateway" cloning system

ccdB is the toxin component of the toxin-antitoxin system of the F plasmid. ccdB is a DNA gyrase inhibitor which causes cell death when the ccdA antitoxin is not present. There is a known mutation of the DNA gyrase gene (gyrA462) that confers resistance to the ccdB toxin. This allows plasmid vectors that contain ccdB to be propagated without associated toxicity in the absence of ccdA. Cloning vectors that contain the ccdB gene can be used to select against vectors that fail to accept a desired insert when transformed into a wild-type gyrA strain. This cloning scheme virtually eliminates background. The Invitrogen "Gateway" cloning system takes advantage of this method [1].

URA3

The URA3 gene from Saccharomyces cerevisiae encodes for orotidine-5'-monophosphate decarboxylase which is involved in de novo synthesis of pyrimidine nucleotides (pathway [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[1]. Many common yeast strains contain a mutation in the URA3 gene making the cells auxotrophic for Uracil. Supplying an intact copy of the URA3 gene on a plasmid or integration cassette restores prototrophy and can be used to select for cells which have taken up the vector containing the marker. URA3 is also known to convert 5-Fluoroorotic acid (5-FOA) into the toxic compound 5-fluorouracil leading to cell death. Therefore, counterselection on 5-FOA will select for clones which have lost the URA3 marker.


It is important to note that the URA3/5-FOA system only works in Saccharomyces cerevisiae in strains with a chromosomal URA3 mutation. Recently the system has been ported to bacterial species[2]. By identifying the URA3 homolog, (pyrF in E.coli and p.putida) and deleting it, URA3 selection/counterseletion can be employed.

pheS

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. When grown on media containing p-chlorophenylalanine cells harboring the mutant version of the gene will be eliminated. Unlike the rpsL counterselection, the mutants pheS phenotype is dominant meaning that the presence of the mutant gene will still cause toxicity when the wild-type version is also present. This strategy has been used in both Gram-negative and Gram-positive bacteria.

References

<biblio>

  1. Boeke1984 pmid=6394957
  2. Galvao2005 pmid=15691944