scholarly journals Translation enhancement by a Dictyostelium gene sequence in Escherichia coli

2019 ◽  
Author(s):  
Tomo Kondo ◽  
Shigehiko Yumura
Keyword(s):  
Escherichia Coli ◽  
Protein Production ◽  
Gene Sequence ◽  
Fluorescent Protein ◽  
Production Systems ◽  
Expression System ◽  
Protein Product ◽  
A Subunit ◽  
Shine Dalgarno Sequence ◽  
Green Fluorescent

AbstractMethods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed “translation enhancement by a Dictyostelium gene sequence” (“TED”) in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3’ end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.

scholarly journals An improved molecular tool for screening bacterial colonies using GFP expression enhanced by a Dictyostelium sequence

BioTechniques ◽  
2020 ◽  
Vol 68 (2) ◽  
pp. 91-95 ◽  
Author(s):  
Tomo Kondo ◽  
Shigehiko Yumura
Keyword(s):  
Escherichia Coli ◽  
Gene Sequence ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Magnetospirillum Gryphiswaldense ◽  
E Coli ◽  
Molecular Tool ◽  
Visual Confirmation ◽  
Green Fluorescent ◽  
User Friendly

During molecular cloning, screening bacterial transformants is a time-consuming and labor-intensive process; however, tractable tools that can be applied to various vectors for visual confirmation of desired colonies are limited. Recently, we reported that translational enhancement by a Dictyostelium gene sequence (TED) boosted protein expression even without an expression inducer in Escherichia coli. Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein enhanced by TED. By inserting a module related to TED into the cloning site in advance, we effectively screened E. coli colonies harboring the desired plasmid functions in a prokaryote ( Magnetospirillum gryphiswaldense) or eukaryote ( Dictyostelium discoideum). Thus, our system represents a user-friendly technique for cloning.

scholarly journals Identification of Promoters for Efficient Gene Expression in Magnetospirillum gryphiswaldense

2009 ◽  
Vol 75 (12) ◽  
pp. 4206-4210 ◽  
Author(s):  
Claus Lang ◽  
Anna Pollithy ◽  
Dirk Schüler
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Expression System ◽  
Antibody Binding ◽  
Magnetotactic Bacterium ◽  
Magnetospirillum Gryphiswaldense ◽  
Efficient Gene ◽  
Green Fluorescent

ABSTRACT To develop an expression system for the magnetotactic bacterium Magnetospirillum gryphiswaldense, we compared gene expression from the widely used Escherichia coli P lac promoter with that from known and predicted genuine M. gryphiswaldense promoters. With the use of green fluorescent protein as a reporter, the highest expression level was observed with the magnetosomal P mamDC promoter. We demonstrate that this promoter can be used for the expression of modified magnetosome proteins to generate “antibody-binding” magnetosomes.

scholarly journals Improving genomically recoded Escherichia coli for the production of proteins containing non-canonical amino acids

2021 ◽  
Author(s):  
Jessica G. Perez ◽  
Erik D. Carlson ◽  
Oliver Weisser ◽  
Camila Kofman ◽  
Kosuke Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Cellular Protein ◽  
Release Factor ◽  
Wild Type ◽  
Genetic Encoding ◽  
Lysine Residues ◽  
Green Fluorescent

AbstractA genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.ΔA) enables efficient genetic encoding of chemically diverse, non-canonical amino acids (ncAAs) into proteins. While C321.ΔA has opened new opportunities in chemical and synthetic biology, this strain has not been optimized for protein production, limiting its utility in widespread industrial and academic applications. To address this limitation, we describe the construction of a series of genomically recoded organisms that are optimized for cellular protein production. We demonstrate that the functional deactivation of nucleases (e.g., rne, endA) and proteases (e.g., lon) increases production of wild-type superfolder green fluorescent protein (sfGFP) and sfGFP containing two ncAAs up to ∼5-fold. Additionally, we introduce a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains. Using an optimized platform, we demonstrated the ability to introduce 2 identical N6-(propargyloxycarbonyl)-L-Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent. We envision that our library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries.

scholarly journals An improved molecular tool for screening bacterial colonies using GFP expression enhanced by a Dictyostelium sequence

2019 ◽  
Author(s):  
Tomo Kondo ◽  
Shigehiko Yumura
Keyword(s):  
Escherichia Coli ◽  
Gene Sequence ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Magnetospirillum Gryphiswaldense ◽  
E Coli ◽  
Molecular Tool ◽  
Visual Confirmation ◽  
Green Fluorescent ◽  
User Friendly

AbstractDuring molecular cloning, screening of bacterial transformants is a time-consuming and labor-intensive process. However, tractable tools that can be applied to various vectors for visual confirmation of desired colonies are limited. Recently, we reported that TED (translational enhancement by a Dictyostelium gene sequence) boosted protein expression even without an expression inducer in Escherichia coli. Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein (GFP) enhanced by TED. By inserting a module related to TED into the cloning site in advance, we effectively screened E. coli colonies harboring the desired plasmid functions in a prokaryote (Magnetospirillum gryphiswaldense) or eukaryote (Dictyostelium discoideum). Thus, our system represents a user-friendly technique for cloning.

scholarly journals Cell-Free Escherichia coli-Based System To Screen for Quorum-Sensing Molecules Interacting with Quorum Receptor Proteins of Streptomyces coelicolor

2009 ◽  
Vol 75 (19) ◽  
pp. 6367-6372 ◽  
Author(s):  
Yung-Hun Yang ◽  
Tae-Wan Kim ◽  
Sung-Hee Park ◽  
Kwangwon Lee ◽  
Hyung-Yeon Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Screening Tool ◽  
Fluorescent Protein ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Expression System ◽  
Homoserine Lactone ◽  
Cell Extract ◽  
Green Fluorescent

ABSTRACT Quorum sensing (QS) is mediated by small molecules and involved in diverse cellular functions, such as virulence, biofilm formation, secondary metabolism, and cell differentiation. In this study, we developed a rapid and effective screening tool based on a cell-free Escherichia coli-based expression system to identify QS molecules of Streptomyces. The binding of QS molecules to γ-butyrolactone receptor ScbR was monitored by changes in the expression levels of the green fluorescent protein reporter in E. coli cell extract. Using this assay system, we could successfully confirm SCB1, a γ-butyrolactone molecule in Streptomyces coelicolor, binding to its known receptor, ScbR. In addition, we have shown that N-hexanoyl-dl-homoserine lactone, one of the QS molecules in many gram-negative bacteria, can regulate ScbR and trigger precocious antibiotic production in S. coelicolor. Our new method can be applied to other strains for which a screening tool for QS molecules has not yet been developed.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

2004 ◽  
Vol 70 (7) ◽  
pp. 3904-3909 ◽  
Author(s):  
Santiago Caballero ◽  
F. Xavier Abad ◽  
Fabienne Loisy ◽  
Françoise S. Le Guyader ◽  
Jean Cohen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Uv Light ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Virus Removal ◽  
Virus Like Particles ◽  
Reverse Transcription Pcr ◽  
Actual Field ◽  
Uv Dose ◽  
Green Fluorescent

ABSTRACT Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

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