Consensus architecture of promoters and transcription units in Escherichia coli: design principles for synthetic biology

2017 ◽  
Vol 13 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Cynthia Rangel-Chavez ◽  
Edgardo Galan-Vasquez ◽  
Agustino Martinez-Antonio
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Design Principles ◽  
Genetic Circuits ◽  
E Coli ◽  
Transcriptional Units

Consensus of the architecture and composition of the elements that form transcriptional units inE. coliand comparison with synthetic genetic circuits.

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 Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

2018 ◽  
Author(s):  
Vanesa Amarelle ◽  
Ananda Sanches-Medeiros ◽  
Rafael Silva-Rocha ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Broad Host Range ◽  
Functional Metagenomics ◽  
Alternative Hosts ◽  
E Coli ◽  
Broad Host Range Plasmid

AbstractAs the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

scholarly journals TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

2019 ◽  
Author(s):  
Felipe A. Millacura ◽  
Mengxi Li ◽  
Marcos Valenzuela-Ortega ◽  
Christopher E. French
Keyword(s):  
Synthetic Biology ◽  
Real World ◽  
Genetically Modified Organisms ◽  
Rna Aptamers ◽  
Genetic Circuit ◽  
Environmental Concerns ◽  
Genetic Circuits ◽  
Transcriptional Units ◽  
Biosensor Applications ◽  
Real World Problems

AbstractWhile synthetic biology represents a promising approach to solve real-world problems, the use of genetically modified organisms is a cause of legal and environmental concerns. Cell-free systems have emerged as a possible solution but much work is needed to optimize their functionality and simplify their usage for Synthetic Biology. Here we present TXO, transcription-only genetic circuits, independent of translation or post-translation maturation. RNA aptamers are used as reaction output allowing the generation of fast, reliable and simple-to-design transcriptional units. TXO cell-free reactions and their possible applications are a promising new tool for fast and simple bench-to-market genetic circuit and biosensor applications.

scholarly journals Development of prokaryotic cell-free systems for synthetic biology

2016 ◽  
Author(s):  
Abel C. Chiao ◽  
Richard M. Murray ◽  
Zachary Z Sun
Keyword(s):  
Synthetic Biology ◽  
Protein Expression ◽  
Genetic Modification ◽  
Natural Phenomena ◽  
Genetic Circuits ◽  
Preparation Methods ◽  
Free System ◽  
Prokaryotic Cell ◽  
Cell Free System ◽  
E Coli

ABSTRACTProkaryotic cell-free systems are currently heavily used for the production of protein that can be otherwise challenging to produce in cells. However, historically cell-free systems were used to explore natural phenomena before the advent of genetic modification and transformation technology. Recently, synthetic biology has seen a resurgence of this historical use of cell-free systems as a prototyping tool of synthetic and natural genetic circuits. For these cell-free systems to be effective prototyping tools, an understanding of cell-free system mechanics must be established that is not purely protein-expression driven. Here we discuss the development ofE. coli-based cell-free systems, with an emphasis on documenting published extract and energy preparation methods into a uniform format. We also discuss additional considerations when applying cell-free systems to synthetic biology.

scholarly journals Sxy Induces a CRP-S Regulon in Escherichia coli

2009 ◽  
Vol 191 (16) ◽  
pp. 5180-5195 ◽  
Author(s):  
Sunita Sinha ◽  
Andrew D. S. Cameron ◽  
Rosemary J. Redfield
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Activation ◽  
Dna Uptake ◽  
Competitive Fitness ◽  
E Coli ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Transcriptional Units ◽  
Sigma H

ABSTRACT Escherichia coli is not considered naturally competent, yet it has homologues of the genes that most competent bacteria use for DNA uptake and processing. In Haemophilus influenzae and Vibrio cholerae, these genes are regulated by the Sxy and cyclic AMP receptor (CRP) proteins. We used microarrays to find out whether similar regulation occurs in E. coli. Expression of sxy strongly induced 63 transcriptional units, 34 of which required CRP for transcriptional activation and had promoter sites resembling the Sxy- and CRP-dependent CRP-S motif previously characterized in H. influenzae. As previously reported, sxy expression also induced the sigma-H regulon. Flagellar operons were downregulated by sxy expression, although motility remained unaffected. The CRP-S regulon included all of E. coli's known competence gene homologues, so we investigated Sxy's effect on competence-associated phenotypes. A sxy knockout reduced both “natural” plasmid transformation and competitive fitness in long-term culture. In addition, expression of plasmid-borne sxy led to production of type IV pilin, the main subunit of the DNA uptake machinery of most bacteria. Although H. influenzae Sxy only weakly activated the E. coli Sxy regulon, induction was dramatically improved when it was coexpressed with its cognate CRP, suggesting that intimate interactions between Sxy and CRP are required for transcriptional activation at CRP-S sites.

scholarly journals Synthetic Biology: Integrated Gene Circuits

Science ◽  
2011 ◽  
Vol 333 (6047) ◽  
pp. 1244-1248 ◽  
Author(s):  
Nagarajan Nandagopal ◽  
Michael B. Elowitz
Keyword(s):  
Synthetic Biology ◽  
Biological Systems ◽  
Design Principles ◽  
Genetic Circuits ◽  
Major Goal ◽  
Gene Circuits ◽  
Biological Design ◽  
Cellular Processes ◽  
Regulatory Architecture ◽  
New Generation

A major goal of synthetic biology is to develop a deeper understanding of biological design principles from the bottom up, by building circuits and studying their behavior in cells. Investigators initially sought to design circuits “from scratch” that functioned as independently as possible from the underlying cellular system. More recently, researchers have begun to develop a new generation of synthetic circuits that integrate more closely with endogenous cellular processes. These approaches are providing fundamental insights into the regulatory architecture, dynamics, and evolution of genetic circuits and enabling new levels of control across diverse biological systems.

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
Author(s):  
Khairun Nessa ◽  
Dilruba Ahmed ◽  
Johirul Islam ◽  
FM Lutful Kabir ◽  
M Anowar Hossain
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Proteinase K ◽  
Microbiology Laboratory ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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