scholarly journals DIRECTED EVOLUTION: SELECTION OF THE HOST ORGANISM

2012 ◽  
Vol 2 (3) ◽  
pp. e201209012 ◽  
Author(s):  
Azadeh Pourmir ◽  
Tyler W. Johannes
Keyword(s):  
Directed Evolution ◽  
Host Organism ◽  
Selection Of

scholarly journals Protein evolution with an expanded genetic code

2008 ◽  
Vol 105 (46) ◽  
pp. 17688-17693 ◽  
Author(s):  
Chang C. Liu ◽  
Antha V. Mack ◽  
Meng-Lin Tsao ◽  
Jeremy H. Mills ◽  
Hyun Soo Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Unnatural Amino Acids ◽  
Selective Advantage ◽  
Display System ◽  
Antibody Library ◽  
Evolution Of Proteins ◽  
Expanded Genetic Code ◽  
Selection Of

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. IV. DIRECTED EVOLUTION OF THE EBG REPRESSOR

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 673-681
Author(s):  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzyme Synthesis ◽  
Regulatory Function ◽  
Wild Type ◽  
Selection For ◽  
Selection Of

ABSTRACT In Escherichia coli, the wild-type repressor of ebg (evolved β-galactosidase) enzyme synthesis, specified by the ebgR  + gene, responds very weakly to lactulose (fructose-β-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 x 10-10. ebgR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

scholarly journals Improving selectivity of DNA-RNA binding zinc finger using directed evolution

2019 ◽  
Author(s):  
Agata Agnieszka Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
Dna Engineering ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates among which, are specific sequences in the double-stranded DNA. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrid using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between modules of the zinc finger. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

scholarly journals Improving selectivity of DNA-RNA binding zinc finger using directed evolution

2019 ◽  
Author(s):  
Agata Agnieszka Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
General Rules ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

scholarly journals Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering

2021 ◽  
Author(s):  
Daisylyn Senna Tan ◽  
Yanpu Chen ◽  
Ya Gao ◽  
Anastasia Bednarz ◽  
Yuanjie Wei ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Phenotypic Selection ◽  
Biochemical Assays ◽  
Genome Wide ◽  
Dna Elements ◽  
Pioneer Factor ◽  
Selection Of

Abstract Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

scholarly journals The Selection of Gamma-Ray Irradiated Higher Yield Rice Mutants by Directed Evolution Method

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1004
Author(s):  
Hiroshi Kato ◽  
Feng Li ◽  
Akemi Shimizu
Keyword(s):  
Directed Evolution ◽  
Gamma Ray ◽  
Nucleotide Polymorphism ◽  
Rice Varieties ◽  
Single Nucleotide ◽  
High Yielding Variety ◽  
Novel Approach ◽  
Yield Trials ◽  
Selection Of ◽  
Plot Design

We have succeeded in selecting four higher yield mutants from five gamma-ray irradiated high-yielding Japanese rice varieties using a novel approach. A total of 464 M2 plants which had heavier total panicle weights per plant were first selected from 9801 irradiated M2 plants. Their higher yields were confirmed by yield trials conducted for three years with a six to ten-pairwise replicated plot design. FukuhibikiH6 and FukuhibikiH8 were selected from an irradiated high-yielding variety Fukuhibiki and showed 1.2% to 22.5% higher yield than their original significantly. YamadawaraH3 was selected from an irradiated high-yielding variety Yamadawara and its yield advantages were 2.7% to 3.9%. However, there was no difference in the genotypes of the 96 SNP (single nucleotide polymorphism) markers between the higher yield mutants and their respective original varieties. The differences in the measured phenotypical traits between each mutant and its original variety were not constant and the actual differences were marginal. Therefore, the higher yields of the selected mutants were likely to have been caused by physiological traits rather than phenotypical traits. The selection method used in this study is an application of the directed evolution method which has long been commonly used in the substantial improvements of microorganisms and their proteins.

scholarly journals Development of Improved Cellulase Variants for the Conversion of Spent Mushroom Substrate Supplemented with Wheat Straw by Directed Evolution

2021 ◽  
Author(s):  
Valentina Mauriello ◽  
Anna Pennacchio ◽  
Irantzu Alegria Dallo ◽  
Laura Garcia Saez ◽  
Petri Ihalainen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Wheat Straw ◽  
Recombinant Expression ◽  
Scale Up ◽  
Wild Type ◽  
Spent Mushroom Substrate ◽  
Glucose Yield ◽  
Hydrolysis Of ◽  
Selection Of

Abstract To improve the Spent mushroom substrate (SMS) saccharification, cloning, recombinant expression in Escherichia coli and characterization of two new GH5 family cellulases (Cel1 and Cel2) were performed. Based on enzymes properties, Cel2 was selected for the generation of 30,000 random mutants by directed evolution in order to develop improved biocatalysts. Error-prone Polymerase Chain Reaction was used for diversity generation in cel2 gene and the screening for activity of mutants allowed selection of 63 improved variants that were subjected to a scale up production. Among these, 13 clones exhibited two-fold higher activity than Cel2 and a higher thermoresistance after 72h. The performances of these mutants in the hydrolysis of pretreated SMS/ wheat straw (40/60) were compared to the wild type Cel2 in conjunction with a commercial enzymatic mixture (MetZyme® SUNO™ BOOSTER 144). All the mutants exhibited a glucose yield two-fold or four fold higher than wild-type Cel2 after 72h of incubation.

scholarly journals Improving selectivity of DNA-RNA binding zinc finger using directed evolution

2019 ◽  
Author(s):  
Agata Agnieszka Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
General Rules ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

scholarly journals CodonGenie: optimised ambiguous codon design tools

2017 ◽  
Author(s):  
Neil Swainston ◽  
Andrew Currin ◽  
Lucy Green ◽  
Rainer Breitling ◽  
Philip J Day ◽  
...  
Keyword(s):  
Amino Acids ◽  
Codon Usage ◽  
Directed Evolution ◽  
Web Application ◽  
Design Tools ◽  
Host Organism ◽  
Stop Codons ◽  
Dna Library ◽  
Protein Mutagenesis

CodonGenie, freely available from http://codon.synbiochem.co.uk, is a simple web application for designing ambiguous codons to support protein mutagenesis applications. Ambiguous codons are derived from specific heterogeneous nucleotide mixtures, which create sequence degeneracy when synthesised in a DNA library. In directed evolution studies, such codons are carefully selected to encode multiple amino acids. For example, the codon NTN, where the code N denotes a mixture of all four nucleotides, will encode a mixture of phenylalanine, leucine, isoleucine, methionine and valine. Given a user-defined target collection of amino acids matched to an intended host organism, CodonGenie designs and analyses all ambiguous codons that encode the required amino acids. The codons are ranked according to their efficiency in encoding the required amino acids while minimising the inclusion of additional amino acids and stop codons. Organism-specific codon usage is also considered.

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