QGEM 2012: Building a Chimeric Flagellar Scaffold for Enhanced Bioremediation and Biosynthesis

2018 ◽  
Author(s):  
Beini Wang ◽  
Kevin Chen
Keyword(s):  
Synthetic Biology ◽  
Metal Binding ◽  
Living Cells ◽  
Genetically Engineered ◽  
Additional Component ◽  
Protein Subunits ◽  
E Coli ◽  
Self Assembling ◽  
Enhanced Bioremediation ◽  
Real World Problems

Synthetic biology is a rapidly expanding field that involves designing biological systems and operating them in living cells. The Queen’s Genetically Engineered Machine (QGEM) team competes annually at the International Genetically Engineered Machine (iGEM) competition, which challenges students around the globe to use synthetic biology to solve real-world problems. Last year, the QGEM team sought to improve the efficiency of bioremediation and biosynthesis by modifying the flagella of E. coli. Flagella are whip-like appendages that many organisms and cells use for locomotion. One flagellum is composed of 20 000 self-assembling protein subunits called flagellin, which can be divided into two parts. One part is necessary to form the flagellum polymer, and the second part is an auxiliary domain with no particular function. This domain can be replaced with functional proteins, such as metal-binding proteins for bioremediation or useful enzymes for biosynthesis. With the incorporation of proteins for binding, adhesion, degradation, and synthesis, normal flagella can be transformed into functional appendages that can be useful in many applications. As an additional component of their project, the QGEM team developed dance as a new means of teaching and explaining science, and incorporated it into their presentation at the iGEM competition.

scholarly journals Students go through the gears at the iGEM competition for engineering biology

2019 ◽  
Vol 41 (3) ◽  
pp. 58-61
Author(s):  
Joshua Lawrence ◽  
Siwat Chang ◽  
Luis Chaves Rodriguez ◽  
Thomas Ouldridge
Keyword(s):  
Synthetic Biology ◽  
Real World ◽  
Genetically Engineered ◽  
Imperial College ◽  
Exciting Opportunity ◽  
Real World Problems

The annual International Genetically Engineered Machine (iGEM) competition, represents an exciting opportunity for students to experience first-hand the potential of synthetic biology approaches to solve real-world problems. In this article, an iGEM team based at Imperial College London share some of the highlights from their participation in the 2018 iGEM event, including sharing their work at the annual Jamboree in Boston, Massachusetts.

scholarly journals Orthogonal translation enables heterologous ribosome engineering in E. coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Natalie S. Kolber ◽  
Ranan Fattal ◽  
Sinisa Bratulic ◽  
Gavriela D. Carver ◽  
Ahmed H. Badran
Keyword(s):  
Synthetic Biology ◽  
16S Rrna ◽  
General Framework ◽  
Living Cells ◽  
Rrna Processing ◽  
Ribosome Engineering ◽  
Subunit Exchange ◽  
E Coli ◽  
Ribosome Binding ◽  
Promising Avenue

AbstractThe ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.

scholarly journals Enhanced Bioaccumulation of Heavy Metal Ions by Bacterial Cells Due to Surface Display of Short Metal Binding Peptides

1999 ◽  
Vol 65 (3) ◽  
pp. 1092-1098 ◽  
Author(s):  
Pavel Kotrba ◽  
Lucie Dolečková ◽  
Víctor de Lorenzo ◽  
Tomas Ruml
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Rational Design ◽  
Binding Capacity ◽  
Surface Display ◽  
Genetically Engineered ◽  
E Coli ◽  
Metal Binding Peptides ◽  
Binding Peptides ◽  
Cell Envelopes

ABSTRACT Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane ofE. coli. Isolated cell envelopes of E. colibearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

2020 ◽  
Vol 14 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Maryam Ahankoub ◽  
Gashtasb Mardani ◽  
Payam Ghasemi-Dehkordi ◽  
Ameneh Mehri-Ghahfarrokhi ◽  
Abbas Doosti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Hazardous Substances ◽  
E Coli ◽  
Spiked Soil ◽  
Engineered Escherichia Coli ◽  
Genetically Manipulated ◽  
Hplc Measurement

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

scholarly journals Refactoring of a synthetic raspberry ketone pathway with EcoFlex

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon J. Moore ◽  
Yonek B. Hleba ◽  
Sarah Bischoff ◽  
David Bell ◽  
Karen M. Polizzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Combinatorial Libraries ◽  
Value Added ◽  
Microtiter Plate ◽  
Fine Tuning ◽  
Golden Gate ◽  
E Coli ◽  
Fine Chemical ◽  
Raspberry Ketone

Abstract Background  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

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 On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
Author(s):  
Xi-Qiang Zhu ◽  
Su-Xia Li ◽  
Hua-Jun He ◽  
Qin-Sheng Yuan
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Mass Ratio ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
Protein Subunits ◽  
E Coli ◽  
Metal Affinity ◽  
Polymerase Chain ◽  
Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

A guide-tag system controlling client enrichment into Y15 peptide-based granules for an in-cell protein recruitment assay

2021 ◽  
Author(s):  
Takayuki Miki ◽  
Masahiro Hashimoto ◽  
Taichi Nakai ◽  
Hisakazu Mihara
Keyword(s):  
Protein Interactions ◽  
Living Cells ◽  
The Self ◽  
Cell Protein ◽  
Protein Protein Interactions ◽  
Self Assembling ◽  
Client Proteins ◽  
Protein Recruitment ◽  
Peptide Tag

A series of guide-tags that can control the enrichment of client proteins into artificial scaffolds constituted by the self-assembling Y15 peptide tag facilitates the analysis of protein–protein interactions in living cells.

scholarly journals Escherichia coli as a genetic tool

1985 ◽  
Vol 95 (3) ◽  
pp. 611-618
Author(s):  
Naomi Datta
Keyword(s):  
Escherichia Coli ◽  
Academic Research ◽  
Genetically Engineered ◽  
Cloning And Expression ◽  
Biological Research ◽  
New Techniques ◽  
E Coli ◽  
Genetic Tool ◽  
The Past ◽  
Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

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