scholarly journals Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria

2008 ◽  
Vol 75 (4) ◽  
pp. 1211-1214 ◽  
Author(s):  
Marie-Pierre Dubeau ◽  
Mariana Gabriela Ghinet ◽  
Pierre-�tienne Jacques ◽  
Nancy Clermont ◽  
Carole Beaulieu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Disruption ◽  
Negative Selection ◽  
Cytosine Deaminase ◽  
Synthetic Gene ◽  
Selection Marker ◽  
Selection System ◽  
Gene Encoding ◽  
Genus Streptomyces ◽  
Negative Selection Marker

ABSTRACT We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors.

scholarly journals A Mutated Cytosine Deaminase Gene, codA (D314A), as an Efficient Negative Selection Marker for Gene Targeting in Rice

2014 ◽  
Vol 55 (3) ◽  
pp. 658-665 ◽  
Author(s):  
Keishi Osakabe ◽  
Ayako Nishizawa-Yokoi ◽  
Namie Ohtsuki ◽  
Yuriko Osakabe ◽  
Seiichi Toki
Keyword(s):  
Gene Targeting ◽  
Negative Selection ◽  
Cytosine Deaminase ◽  
Selection Marker ◽  
Negative Selection Marker ◽  
Cytosine Deaminase Gene

Expression of a synthetic gene encoding human transthyretin in Escherichia coli

2003 ◽  
Vol 30 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Kimiaki Matsubara ◽  
Mineyuki Mizuguchi ◽  
Keiichi Kawano
Keyword(s):  
Escherichia Coli ◽  
Synthetic Gene ◽  
Gene Encoding

scholarly journals CD26: A negative selection marker for human Treg cells

2012 ◽  
Vol 81A (10) ◽  
pp. 843-855 ◽  
Author(s):  
Francisco J. Salgado ◽  
Amparo Pérez-Díaz ◽  
Nora M. Villanueva ◽  
Olaya Lamas ◽  
Pilar Arias ◽  
...  
Keyword(s):  
Negative Selection ◽  
Treg Cells ◽  
Selection Marker ◽  
Negative Selection Marker

scholarly journals Strategy for Designing the Synthetic Gene Encoding Human papillomavirus Major Capsid L1 Protein for Heterologous Expression in Escherichia coli System

2020 ◽  
Vol 8 (2) ◽  
pp. 225
Author(s):  
Kartika Sari Dewi ◽  
Wien Kusharyoto
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Codon Optimization ◽  
Gc Content ◽  
Web Server ◽  
Synthetic Gene ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Construct ◽  
L1 Protein

DNA is widely used to construct heterologously expressed genes. The adaptation of the codons to the host organism is necessary in order to ensure sufficient production of proteins. The GC content, codon identity and the mRNA from the translation site are also important in the design of the gene construct. This study performed a strategy for the design of synthetic gene encoding HPV52 L1 protein and several analyses at the genetic level to optimize its protein expression in the Escherichia coli BL21(DE3) host. The determination of the codon optimization was performed by collecting 75 HPV52 L1 protein sequences in the NCBI database. Furthermore, all the sequences were analyzed using multiple global alignments by Clustal Omega web server. Once the model was determined, codon optimization was performed using OPTIMIZER and the web server of the IDT codon optimization tool based on the E. Coli B. The generated open reading frame (ORF) sequence was analyzed using Restriction mapper web server to choose the restriction site for facilitating the cloning stage, which is adjusted for pJExpress414 expression vector. To maximize the protein expression level, the mRNA secondary structure analysis around the ribosome binding site (rbs) was performed. A slight modification at the 5’-terminal end waa carried out in order to get more accessible rbs and increasing mRNA folding free energy. Finally, the construction of the synthetic gene was confirmed to ensure that no mutation occurs in the protein and to calculate its Codon Adaptation Index (CAI) and GC content. The above strategy, which leads to a good ORF sequence with the value of the free mRNA folding energy around rbs, is -5.5 kcal / mol, CAI = 0.787 and GC content 49.5%. This result is much better than its original gene. This result is much better compared to its native gene. Theoretically it is possible that this synthetic gene construct generates a high level protein expression in E. coli BL21 (DE3) under the regulation of the T7 promoter.

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