scholarly journals Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

2021 ◽  
Author(s):  
Michela Notarangelo ◽  
Alessandro Quattrone ◽  
Massimo Pizzato ◽  
Sheref S. Mansy ◽  
O. Duhan Toparlak
Keyword(s):  
Room Temperature ◽  
Colorimetric Detection ◽  
In Vitro Transcription ◽  
Viral Rna ◽  
Rna Sequences ◽  
Rna Detection ◽  
E Coli ◽  
Free Gene ◽  
Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

Fluorescent primer-based in vitro transcription system of viral RNA-dependent RNA polymerases

2013 ◽  
Vol 433 (2) ◽  
pp. 92-94
Author(s):  
Qiang Wang ◽  
Leiyun Weng ◽  
Hongbing Jiang ◽  
Shijian Zhang ◽  
Tetsuya Toyoda
Keyword(s):  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Viral Rna ◽  
Transcription System

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Ahlquist ◽  
M Janda
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
In Vitro Translation ◽  
In Vitro Production ◽  
Genomic Rnas

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

scholarly journals Structural basis for transcription inhibition by E. coli SspA

2020 ◽  
Vol 48 (17) ◽  
pp. 9931-9942 ◽  
Author(s):  
Fulin Wang ◽  
Jing Shi ◽  
Dingwei He ◽  
Bei Tong ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Acid Tolerance ◽  
In Vitro Transcription ◽  
Nucleotide Metabolism ◽  
Structural Basis ◽  
E Coli ◽  
Promoter Escape ◽  
Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

Functional and structural differences between photosynthetic and heterotrophic Rhodospirillum rubrum ribosomes and S-100 fractions

1976 ◽  
Vol 22 (10) ◽  
pp. 1522-1539 ◽  
Author(s):  
C. T. Chow
Keyword(s):  
Rhodospirillum Rubrum ◽  
Photosynthetic Pigment ◽  
Free Protein ◽  
E Coli ◽  
Highly Active ◽  
Structural Differences ◽  
S 100 ◽  
Cell Free Protein Synthesis ◽  
Fraction Addition

Cell-free, protein-synthesizing activity has been tested by using various combinations of the S-100 and ribosome fractions prepared from photosynthetic and heterotrophic Rhodospirillum rubrum. The photosynthetic ribosomes are highly active when combined with either the photosynthetic or the heterotrophic S-100 fractions, whereas the heterotrophic ribosomes are active only when combined with the photosynthetic S-100 fraction. Addition of a photosynthetic pigment-containing fraction to the homologous heterotrophic system is, however, able to stimulate its activity. An inhibitor and an activator involved in cell-free protein synthesis have been isolated from the stationary heterotrophic cells. The inhibitor is a very small, dialyzable compound which inhibits not only the R. rubrum but also the E. coli protein-synthesizing activity in vitro, whereas the activator is a non-dialyzable, small RNA molecule capable of stimulating only the R. rubrum activity. Differences exist between the photosynthetic and the heterotrophic systems in their response to various chemical compounds and to light as well as in their structure.

Vibrio harveyi RNA polymerase: purification and resolution from gyrase A

1992 ◽  
Vol 70 (8) ◽  
pp. 698-702 ◽  
Author(s):  
Elana Swartzman ◽  
Edward A. Meighen
Keyword(s):  
Rna Polymerase ◽  
Topoisomerase Ii ◽  
Vibrio Harveyi ◽  
In Vitro Transcription ◽  
E Coli ◽  
Gyrase A ◽  
A Subunit ◽  
Terminal Amino ◽  
Promoter Specificity

RNA polymerase was purified from Vibrio harveyi and found to contain polypeptides (β,β′, α, and σ) closely corresponding to those of the Escherichia coli enzyme. In vitro transcription studies using V. harveyi and E. coli RNA polymerase demonstrated that the purified V. harveyi RNA polymerase is functional and that the two enzymes have the same promoter specificity. Chromatography through a monoQ column was required to remove a 100-kilodalton protein that was present in large amounts and copurified with the RNA polymerase. N-terminal amino acid sequencing showed that the first 18 amino acids of the 100-kilodalton protein shares 78% sequence identity with the A subunit of gyrase or topoisomerase II. The abundance of the gyrase A protein is unprecedented and may be linked to bioluminescence.Key words: Vibrio harveyi, RNA polymerase, gyrase, bioluminescence.

scholarly journals Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Atsuko Uyeda ◽  
Takayoshi Watanabe ◽  
Takahiro Hohsaka ◽  
Tomoaki Matsuura
Keyword(s):  
Lipid Bilayer ◽  
Lipid Membrane ◽  
Covalent Bond ◽  
In Vitro Transcription ◽  
Translation System ◽  
Simple Method ◽  
Outer Leaflet ◽  
Liposome Surface ◽  
Cell Free Protein Synthesis

Abstract Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different proteins are found on the inner and outer leaflets of the membrane, and they are essential for various biochemical reactions, including those related to signal transduction and cell morphology. While in vitro lipid bilayer reconstitution with asymmetric phospholipid compositions has been reported, the reconstitution of lipid bilayer where different proteins are localized in the inner and outer leaflet, thereby enables asymmetric protein localizations, has remained difficult. Herein, we developed a simple method to achieve this asymmetry using an in vitro transcription–translation system (IVTT). The method used a benzylguanine (BG) derivative-modified phospholipid, which forms a covalent bond with a snap-tag sequence. We show that purified snap-tagged protein can be localized to the cell-sized liposome surface via an interaction between BG and the snap-tag. We then show that IVTT-synthesized proteins can be located at the lipid membrane and that different proteins can be asymmetrically localized on the outer and inner leaflets of liposomes.

scholarly journals Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations

2001 ◽  
Vol 183 (7) ◽  
pp. 2289-2297 ◽  
Author(s):  
Marco P. Cicero ◽  
Meghan M. Sharp ◽  
Carol A. Gross ◽  
Kenneth N. Kreuzer
Keyword(s):  
Rna Polymerase ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Positive Control ◽  
Plaque Morphology ◽  
Mutant Proteins ◽  
E Coli

ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. AmotA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. colipromoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

scholarly journals Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

1969 ◽  
Vol 115 (3) ◽  
pp. 353-361 ◽  
Author(s):  
John O. Bishop ◽  
Forbes W. Robertson
Keyword(s):  
Rna Polymerase ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Bacteriophage T4 ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
E Coli ◽  
New Methods

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

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