Construction of a Positive Selection Marker by a Lethal Gene with the Amber Stop Codon(s) Regulator

2006 ◽  
Vol 70 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Sumiko OHASHI-KUNIHIRO ◽  
Hiroko HAGIWARA ◽  
Masafumi YOHDA ◽  
Haruhiko MASAKI ◽  
Masayuki MACHIDA
Keyword(s):  
Positive Selection ◽  
Stop Codon ◽  
Selection Marker ◽  
Lethal Gene ◽  
Amber Stop Codon

scholarly journals Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 546 ◽  
Author(s):  
David Schwark ◽  
Margaret Schmitt ◽  
John Fisk
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Codon Reassignment ◽  
Methanocaldococcus Jannaschii ◽  
E Coli ◽  
Amber Stop Codon ◽  
Orthogonal Pair ◽  
Amber Codon ◽  
Quantitative Contribution ◽  
Stop Codon Reassignment

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

scholarly journals Exploiting a Natural Auxotrophy for Genetic Selection

2012 ◽  
Vol 78 (16) ◽  
pp. 5926-5928 ◽  
Author(s):  
Elizabeth Ramage ◽  
Larry Gallagher ◽  
Colin Manoil
Keyword(s):  
Escherichia Coli ◽  
Positive Selection ◽  
Growth Medium ◽  
Genetic Selection ◽  
Selection Marker ◽  
Content Type ◽  
Francisella Novicida ◽  
Shuttle Plasmid ◽  
Histidinol Dehydrogenase ◽  
Highly Virulent

ABSTRACTWe exploited the natural histidine auxotrophy ofFrancisellaspecies to develophisD(encodes histidinol dehydrogenase) as a positive selection marker. A shuttle plasmid (pBR103) carryingEscherichia coli hisDand designed for cloning of PCR fragments replicated in both attenuated and highly virulentFrancisellastrains. During this work, we formulated a simplified defined growth medium forFrancisella novicida.

Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae

2001 ◽  
Vol 305 (4) ◽  
pp. 703-714 ◽  
Author(s):  
Thomas Winckler ◽  
Christine Trautwein ◽  
Christina Tschepke ◽  
Christin Neuhäuser ◽  
Ilse Zündorf ◽  
...  
Keyword(s):  
Gene Function ◽  
Growth And Development ◽  
Nuclear Protein ◽  
Stop Codon ◽  
Function Analysis ◽  
Amber Stop Codon ◽  
Gene Function Analysis
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