Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter

1993 ◽  
Vol 41 (9) ◽  
pp. 837-845 ◽  
Author(s):  
Nikos C. Pagratis ◽  
Helen R. Revel
Keyword(s):  
Rna Polymerase ◽  
Host Range ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Broad Host Range ◽  
Regulated Expression

scholarly journals Engineering efficient termination of bacteriophage T7 RNA polymerase transcription

2021 ◽  
Author(s):  
Diana Gabriela Calvopina Chavez ◽  
Mikaela Anne Gardner ◽  
Joel S Griffitts
Keyword(s):  
Rna Polymerase ◽  
Molecular Level ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Lower Efficiency ◽  
Transcriptional Termination ◽  
T7 Polymerase

The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

scholarly journals Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

1987 ◽  
Vol 7 (7) ◽  
pp. 2538-2544 ◽  
Author(s):  
T R Fuerst ◽  
P L Earl ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Target Gene ◽  
Target Genes ◽  
Expression System ◽  
Recombinant Vaccinia Virus ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Virus Surface ◽  
Recombinant Vaccinia

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes

1987 ◽  
Vol 7 (7) ◽  
pp. 2538-2544
Author(s):  
T R Fuerst ◽  
P L Earl ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Target Gene ◽  
Target Genes ◽  
Expression System ◽  
Recombinant Vaccinia Virus ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Virus Surface ◽  
Recombinant Vaccinia

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.

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