scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Author(s):  
Changhao Bi ◽  
Bin Xiong ◽  
Muzi Hu ◽  
Zhongkang Li ◽  
Li Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
T7 Promoter ◽  
T7 Expression System ◽  
Plasmid Size ◽  
Rna Polymerase Gene ◽  
Essential Sequence ◽  
Electroporation Efficiency

Abstract Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for the metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression. Although current plasmid systems for R. eutropha provide a basic platform for gene expression, the performance of the induction systems is still limited. In addition, the sizes of the cloned genes is limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into genome of R. eutropha, and cloning the T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted, reducing the expression plasmid size to 3392 bp, and the electroporation efficiency was improved 4 times. As a result, the highest expression level of Rfp was enhanced slightly, and the L-arabinose concentration necessary for induction was decreased 20 times. Conclusions: R. eutropha with the T7 expression system provides an efficient platform for protein expression and synthetic biology applications.

scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Author(s):  
Muzi Hu ◽  
Bin Xiong ◽  
Zhongkang Li ◽  
Li Liu ◽  
Siwei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Production ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
Expression Plasmid ◽  
T7 Promoter ◽  
T7 Expression System ◽  
Plasmid Size ◽  
Rna Polymerase Gene ◽  
Electroporation Efficiency

Abstract Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Author(s):  
Muzi Hu ◽  
Bin Xiong ◽  
Zhongkang Li ◽  
Li Liu ◽  
Siwei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Production ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
Expression Plasmid ◽  
T7 Promoter ◽  
T7 Expression System ◽  
Plasmid Size ◽  
Rna Polymerase Gene ◽  
Electroporation Efficiency

Abstract Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

scholarly journals Tightly Regulated Gene Expression System in Salmonella enterica Serovar Typhimurium

2002 ◽  
Vol 184 (21) ◽  
pp. 6056-6059 ◽  
Author(s):  
Jeffrey McKinney ◽  
Cecilia Guerrier-Takada ◽  
Jorge Galán ◽  
Sidney Altman
Keyword(s):  
Gene Expression ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Expression System ◽  
Dynamic Control ◽  
Control Element ◽  
T7 Promoter ◽  
Serovar Typhimurium ◽  
Regulated Gene Expression ◽  
Rna Polymerase Gene

ABSTRACT A new Salmonella enterica serovar Typhimurium strain has been constructed to facilitate tightly regulated gene expression. Arabinose-inducible and glucose-repressible expression of a T7 RNA polymerase gene that has been integrated with an adjacent araC-PBAD control element into the bacterial chromosome allows dynamic control of T7 promoter-driven RNA transcription.

scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Muzi Hu ◽  
Bin Xiong ◽  
Zhongkang Li ◽  
Li Liu ◽  
Siwei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
T7 Promoter ◽  
Gene Expression System ◽  
Novel Gene

A T7 promoter-specific, inducible protein expression system for

1996 ◽  
Vol 250 (2) ◽  
pp. 230 ◽  
Author(s):  
Birgit Conrad ◽  
R. S. Savchenko ◽  
Roland Breves ◽  
J. Hofemeister
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
T7 Promoter ◽  
Protein Expression System ◽  
Inducible Protein

scholarly journals Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

2007 ◽  
Vol 73 (15) ◽  
pp. 4849-4856 ◽  
Author(s):  
Ryoma Miyake ◽  
Jun Kawamoto ◽  
Yun-Lin Wei ◽  
Masanari Kitagawa ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Low Temperature ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Maximum Yield ◽  
Broad Host Range ◽  
Psychrophilic Bacterium ◽  
T7 Promoter ◽  
Cold Adapted ◽  
Protein Expression System

ABSTRACT A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.

scholarly journals Enhancement of transient erythropoietin protein expression by valproic acid in CHO‐K1 suspension adapted cells

2020 ◽  
Vol 25 (1) ◽  
pp. 28
Author(s):  
Yana Rubiyana ◽  
Retno Damajanti Soejoedono ◽  
Adi Santoso
Keyword(s):  
Gene Expression ◽  
Valproic Acid ◽  
Protein Expression ◽  
Histone Deacetylase Inhibitors ◽  
Expression System ◽  
Transient Gene Expression ◽  
Optimum Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stable Gene ◽  
Gene Expression System

Erythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells.

CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

2021 ◽  
Author(s):  
Changchuan Ye ◽  
Xi Chen ◽  
Mengjie Yang ◽  
Xiangfang Zeng ◽  
Shiyan Qiao
Keyword(s):  
Rna Polymerase ◽  
Mutant Strain ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacterial Strains ◽  
T7 Promoter ◽  
E Coli ◽  
T7 Expression System ◽  
High Level

Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

scholarly journals Efficient Expression of Soluble Recombinant Protein Fused with Core-Streptavidin in Bacterial Strain with T7 Expression System

2020 ◽  
Vol 3 (4) ◽  
pp. 82
Author(s):  
Ammar Tarar ◽  
Esmael M. Alyami ◽  
Ching-An Peng
Keyword(s):  
Fusion Protein ◽  
Thymidine Phosphorylase ◽  
Expression System ◽  
Model System ◽  
Carcinoma Cells ◽  
T7 Promoter ◽  
E Coli ◽  
Efficient Expression ◽  
T7 Expression System ◽  
Soluble Recombinant Protein

The limited amount of fusion protein transported into cytosol milieu has made it challenging to obtain a sufficient amount for further applications. To avoid the laborious and expensive task, T7 promoter-driving pET-30a(+) coding for chimeric gene of thymidine phosphorylase and core streptavidin as a model system was constructed and transformed into a variety of E. coli strains with T7 expression system. Our results demonstrated that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system is able to provide efficient expression of soluble TP-coreSA fusion protein for purification. Moreover, the eluted TP-coreSA fusion protein tethered on biotinylated A549 carcinoma cells could effectively eliminate these malignant cells after administrating prodrug 5′-DFUR.

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