Auxiliary Module Promotes the Synthesis of Carboxysomes in E. coli to Achieve High-Efficiency CO2 Assimilation

2021 ◽  
Author(s):  
Yuwei Zhang ◽  
Juan Zhou ◽  
Yuchen Zhang ◽  
Tiangang Liu ◽  
Xiaoyun Lu ◽  
...  
Keyword(s):  
High Efficiency ◽  
Co2 Assimilation ◽  
E Coli ◽  
Auxiliary Module

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

scholarly journals Improved CUT&RUN chromatin profiling and analysis tools

2019 ◽  
Author(s):  
Michael P. Meers ◽  
Terri Bryson ◽  
Steven Henikoff
Keyword(s):  
Fusion Protein ◽  
High Efficiency ◽  
Low Cost ◽  
Protein A ◽  
Micrococcal Nuclease ◽  
Permeabilized Cells ◽  
E Coli ◽  
Carry Over ◽  
Chromatin Profiling ◽  
Premature Release

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

scholarly journals Leveraging modern DNA assembly techniques for rapid, markerless genome modification

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Ilya B Tikh ◽  
James C Samuelson
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Host Cells ◽  
Dna Assembly ◽  
E Coli ◽  
Recombination Efficiency ◽  
Genome Modification ◽  
Counter Selection ◽  
K 12 ◽  
Polymerase Chain Reaction Primers

Abstract The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
Author(s):  
Peter L. Wang
Keyword(s):  
Directed Evolution ◽  
High Efficiency ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Strand Breaks ◽  
E Coli ◽  
Hybrid Genes ◽  
Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

scholarly journals Efficient Transformation System forPropionibacterium freudenreichii Based on a Novel Vector

2001 ◽  
Vol 67 (2) ◽  
pp. 499-503 ◽  
Author(s):  
J. P. M. Jore ◽  
N. van Luijk ◽  
R. G. M. Luiten ◽  
M. J. van der Werf ◽  
P. H. Pouwels
Keyword(s):  
High Efficiency ◽  
Propionibacterium Freudenreichii ◽  
Transformation System ◽  
Erythromycin Resistance ◽  
E Coli ◽  
Recognition Sites ◽  
Dna Ligation ◽  
Enzyme Recognition ◽  
Restriction Modification ◽  
Vector Dna

ABSTRACT A 3.6-kb endogenous plasmid was isolated from aPropionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of anEscherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli andP. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacteriumtransformant dramatically increased the efficiency of transformation (≥108 colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.

scholarly journals Development of PAR-CLIP to analyze RNA-protein interactions in prokaryotes

2019 ◽  
Author(s):  
Sandeep Ojha ◽  
Chaitanya Jain
Keyword(s):  
Protein Interactions ◽  
Messenger Rna ◽  
High Efficiency ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleoside Analogs ◽  
E Coli ◽  
Rna Targets ◽  
Genome Wide ◽  
Established Technique

AbstractThe ability to identify RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as “Crosslinking and Immunoprecipitation” (CLIP) has revolutionized the genome-wide discovery of RNA targets. Among the different versions of CLIP developed, the incorporation of photoactivable nucleoside analogs into cellular RNA has proven to be especially valuable, allowing for high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP). Although PAR-CLIP has become an established technique for use in eukaryotes, it has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we first investigated whether 4-thiouridine (4SU), a photoactivable nucleoside, can be incorporated into E. coli RNA. After determining 4SU incorporation into RNA, we developed suitable conditions for crosslinking of proteins in E. coli cells and for the isolation of crosslinked RNA. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA) - messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq. Based on our results, PAR-CLIP represents an improved method to identify the RNAs recognized by RNA-BPs in prokaryotes.

scholarly journals Selection of the optimal composition of vegetable oil and chlorophyllipt oil components

2019 ◽  
Vol 2 (3) ◽  
pp. 11-14
Author(s):  
R. M. Sachuk ◽  
Ya. S. Stravsky ◽  
YU. V. Horyuk ◽  
O. A. Katsaraba ◽  
S. V. Zhyhalyuk
Keyword(s):  
Essential Oil ◽  
Essential Oils ◽  
High Efficiency ◽  
Skin Diseases ◽  
Antimicrobial Properties ◽  
Optimal Composition ◽  
Tea Tree ◽  
E Coli ◽  
Siberian Pine ◽  
Oily Solution

Investigate the antimicrobial properties of various concentrations of vegetable essential oils in combination with an oil solution of chlorophyllipt as possible ingredients for ointments for wounds. Microbiological tests were performed according to standard methods using E. coli test cultures, S. aureus, Str. agalactiae and P. fluorescens. The results of studies of antibacterial activity of both individual ingredients and their combinations are presented, namely: 4 % essential oil of Siberian pine, 2 % essential oil of eucalyptus, 1.5 % essential oil of cloves, 1.5 % of essential oil of cedar, 2.0 % tea tree essential oil and 1.0 % chlorophyllipt oil solution. Bacteria, which are usually typical agents of wound infections, have been found to be quite sensitive to the drugs tested. High activity of essential oils and oily solution of chlorophyllipt with respect to E. coli and S. aureus was revealed. In particular, a 1.0 % oily solution of chlorophyllipt caused staphylococcal growth retardation zones whose diameters were 1.4 times larger than the antibiotic clindamycin. It was determined that representatives of gram-negative microflora were more sensitive to the investigated essential oils and chlorophyll. The optimal composition of the experimental drug called “Ointment for wounds” is offered. The results of preclinical testing showed a sufficiently high efficiency compared to traditional means. The results obtained with regard to antimicrobial activity indicate the prospect of using preparations based on the essential oil of Siberian pine, eucalyptus, carnation, cedar, tea tree and oil solution of chlorophyllipt for the treatment of skin diseases in animals. This data will help to develop new effective and safe veterinary treatments for wound care.

Assessment of Signal Peptides to Optimize Interleukin 2 (IL-2) Folding and Expression

2019 ◽  
Vol 16 (3) ◽  
pp. 188-198 ◽  
Author(s):  
Narges Nazifi ◽  
Mojtaba Tahmoorespur ◽  
Mohammad Hadi Sekhavati ◽  
Alireza Haghparast ◽  
Mohammad Ali Behroozikhah
Keyword(s):  
Cellular Localization ◽  
High Efficiency ◽  
Low Cost ◽  
Expression System ◽  
Interleukin 2 ◽  
Signal Peptides ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Secretion Pathway ◽  
Physico Chemical

Background:Using a bacterial expression system such as Escherichia coli (E. coli) is very common for protein expression because of its simplicity, low cost and high efficiency.Objective:In order to express proteins that contain di-sulfide bands, an oxidative space such as the periplasmic environment of the bacteria is required. Therefore, a leader sequence which named Signal Peptide (SP) is needed to direct recombinant protein to fold in periplasmic space. Interleukin-2 (IL-2) is a prominent cytokine which known as growth factor for T-cells and typically produced by a variety of immune cells that stimulate and regulate inflammatory and immune responses.Methods:This study was designed to predict the best signal peptides to express IL-2 in E. coli. To predict the best signal peptides for the expression of IL-2 in Gram-negative bacteria (E. coli), forty-five sequences of SPs were extracted from data base. Some most important details such as n, h and c regions of signal peptides and their probability were studied through the signalP software. </P><P> Afterwards, physico–chemical features of SPs were analyzed by Portparam and Solpro tools. Finally, secretion-pathway and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB softwares.Results:At the end of the in-silico analyzes, it was determined that ccmH, PelB, traU, yohN, lolA, yhcN are the most reliable SPs, respectively, with highest score and best performing to express the IL-2 protein in E. coli.

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