A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator

Gene ◽  
1994 ◽  
Vol 142 (1) ◽  
pp. 61-66 ◽  
Author(s):  
Marina I. Lebedeva ◽  
Ekaterina V. Rogozhkina ◽  
Nikolay A. Tsyba ◽  
Sergey V. Mashko
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Recombinant Plasmid ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
T7 Promoter ◽  
Lac Operator

scholarly journals Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

2020 ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
Chemical Production ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Shock Proteins

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Results Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At first, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Conclusion Inducer and lacI/lacO regulators on chromosome and plasmid have been investigated in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory in the future.

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

2021 ◽  
Vol 15 (1) ◽  
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

AbstractT7 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.

Development of an inducible T7 expression system in Bacillus subtilis ATCC 6051a for production of α-L-arabinofuranosidase

2020 ◽  
Author(s):  
Minghua Ji ◽  
Sijie Li ◽  
Yunhui Liu ◽  
Haiying Wu ◽  
Qiao Chen ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Fermentation Broth ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
T7 Promoter ◽  
Subtilis Atcc ◽  
Bacillus Subtilis Atcc

Abstract Background: Xylan is the second most abundant polysaccharide biomass on the earth, the polymers have a backbone of β-1,4-linked xylose residues with various side-chain substitutions, such as arabinose, acetic acid, glucuronic acid, and other esterified groups, thus, the removal of arabinose side groups by α-L-arabinofuranosidases is helpful in various industrial processes involving xylan treatment. Bacillus subtilis ATCC 6051a is known for its excellent capacity of secretory production of recombinant peptides, however, poor experience in genetic manipulation and lack of universal expression elements impede this strain for wider application.Results: Xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, and the transformation efficiency of the engineered strain B. subtilis 164S was increased by more than 1000 folds. B. subtilis 164S was further modified to generate B. subtilis 164T7P which incorporates a D-xylose inducible T7 RNA polymerase. The recombinant GFP expressed by 164T7P is more than thirteen times that achieved by P43 promoter, representing the most efficient expression system that had been ever reported in B. subtilis . Subsequently, abfA1 , encoding a glycoside hydrolase (GH) family 51 enzyme was cloned and overexpressed in 164T7P. The activity of recombinant α-L-arabinofuranosidase (AbfA1) reached 90.6±2.0 U mL -1 in the fermentation broth. Using p NPA as a substrate, kinetic parameters of the crude enzyme were Km of 1.4±0.1 mM and kcat of 139.4 s −1 . The optimum temperature and pH of the recombinant AbfA1 towards p NPA were observed to be 45 °C and pH 6.5, respectively.Conclusion: With enhanced cellular competence and introduction of T7 RNA polymerase, B. subtilis ATCC 6051a was engineered as a versatile cell tool for recombinant production of heterologous peptides employing T7 promoter. The novel expression kit demonstrated a very low level of leaky expression of target genes. The efficiency and applicability of the system were demonstrated by high-level production of a bacterial type α-L-arabinofuranosidase.

scholarly journals Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
T7 Promoter ◽  
Iptg Induction ◽  
E Coli ◽  
A Cell

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

scholarly journals Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter/RNA polymerase expression system.

1993 ◽  
Vol 40 (2) ◽  
pp. 273-278
Author(s):  
J Nieradko ◽  
B Podgórska
Keyword(s):  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Expression System ◽  
Inhibitory Effect ◽  
High Rate ◽  
T7 Rna Polymerase ◽  
T7 Promoter ◽  
System A ◽  
Expression Of Genes ◽  
Strong Inhibitory Effect

A fragment of T4 DNA (XbaI-HindIII) comprising the genes 51, 27, 28, which encodes the central plug proteins was cloned into plasmid pT7-5 and p7-6 (T7 RNA polymerase expressing system). The examined genes were only overexpressed when the orientation of cloned DNA to promoter phi 10 was as follows: promoter phi 10 and genes 51, 27, 28. This was achieved when the fragment (XbaI-HindIII) was cloned into plasmid pT7-5. Gene 27 and 28 were overexpressed when the intact fragment (XbaI-HindIII) was used. The high rate of the synthesis of proteins 27 and/or 28 had a strong inhibitory effect on the level of synthesis of the product of gene 51. For the overexpression of gene 51 in this system a deletion derivate which was devoid of gene 28 and a larger fragment of gene 27 was prepared.

Novel expression system for Corynebacterium acetoacidophilum and Escherichia coli based on the T7 RNA polymerase-dependent promoter

2013 ◽  
Vol 97 (17) ◽  
pp. 7755-7766 ◽  
Author(s):  
Md. Javed Equbal ◽  
Preeti Srivastava ◽  
Gopal Prasad Agarwal ◽  
Jahar Kanti Deb
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Expression System ◽  
T7 Rna Polymerase
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