Synthetic Biology with an All E. coli TXTL System: Quantitative Characterization of Regulatory Elements and Gene Circuits

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

Download Full-text

GreA and GreB enhanceEscherichia coliRNA polymerase transcription rate in a reconstituted transcription-translation system

10.1101/024604 ◽

2015 ◽

Author(s):

Lea L. De Maddalena ◽

Henrike Niederholtmeyer ◽

Matti Turtola ◽

Zoe Swank ◽

Georgiy A. Belogurov ◽

...

Keyword(s):

Synthetic Biology ◽

Rna Polymerase ◽

Sigma Factor ◽

Expression System ◽

Genetic Networks ◽

Translation System ◽

Transcription Rate ◽

Pure System ◽

E Coli

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate ofEscherichia coliRNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates ofE. coliRNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of naturalE. colipromoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and syntheticE. colipromoters and characterization of their regulation in minimal and defined reaction conditions making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.

Download Full-text

Genome-wide Functional Characterization of Escherichia coli Promoters and Regulatory Elements Responsible for their Function

10.1101/2020.01.04.894907 ◽

2020 ◽

Cited By ~ 8

Author(s):

Guillaume Urtecho ◽

Kimberly D. Insigne ◽

Arielle D. Tripp ◽

Marcia Brinck ◽

Nathan B. Lubock ◽

...

Keyword(s):

Promoter Activity ◽

Functional Characterization ◽

Regulatory Elements ◽

Machine Learning Algorithms ◽

Regulatory Sequences ◽

Primary Sequence ◽

Entire Genome ◽

E Coli ◽

Full Characterization

SummaryDespite decades of intense genetic, biochemical, and evolutionary characterizations of bacterial promoters, we still lack the basic ability to identify or predict transcriptional activities of promoters using primary sequence. Even in simple, well-characterized organisms such as E. coli there is little agreement on the number, location, and strength of promoters. Here, we use a genomically-encoded massively parallel reporter assay to perform the first full characterization of autonomous promoter activity across the E. coli genome. We measure promoter activity of >300,000 sequences spanning the entire genome and precisely map 2,228 promoters active in rich media. We show that antisense promoters have a profound effect on global transcription and how codon usage has adapted to encode intragenic promoters. Furthermore, we perform a scanning mutagenesis of 2,057 promoters to uncover regulatory sequences responsible for regulating promoter activity. Finally, we show that despite these large datasets and modern machine learning algorithms, the task of predicting promoter activity from primary sequence sequence is still challenging.

Download Full-text

The all-E. Coli TXTL Toolbox 3.0: New Capabilities of a Cell-Free Synthetic Biology Platform

Synthetic Biology ◽

10.1093/synbio/ysab017 ◽

2021 ◽

Author(s):

David Garenne ◽

Seth Thompson ◽

Amaury Brisson ◽

Aset Khakimzhan ◽

Vincent Noireaux

Keyword(s):

Synthetic Biology ◽

Expression System ◽

Building Blocks ◽

Regulatory Elements ◽

Free Expression ◽

Batch Mode ◽

E Coli ◽

A Genome ◽

Synthetic Cells

Abstract The new generation of cell-free gene expression systems enables prototyping and engineering biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription-translation (TXTL) platforms remains a major goal to either execute larger DNA programs, or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch mode reactions, synthesis of the fluorescent reporter protein eGFP reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced to concentrations of more than 8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kbp and about 60 genes, is produced at a concentration of 1013 PFU/ml. This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for either testing regulatory elements and circuits, biomanufacturing biologics, or building synthetic cells.

Download Full-text

MATERIAL PROPERTIES ANALYSIS OF CIRCUIT SUBASSEMBLIES: QUANTITATIVE CHARACTERIZATION OF SAPPHIRE (alpha-Al203) AND SILICON NITRIDE (Si3N4) USING CRYOGENIC CYCLING

10.1615/tfec2019.ref.027456 ◽

2019 ◽

Author(s):

Kirsten A. Lovelace ◽

Sonya T. Smith

Keyword(s):

Silicon Nitride ◽

Material Properties ◽

Quantitative Characterization

Download Full-text

Quantitative characterization of keyhole absorption mechanisms in 20 kW-class CO2 laser welding processes

International Congress on Applications of Lasers & Electro-Optics ◽

10.2351/1.5066122 ◽

2002 ◽

Author(s):

Jay F. Tu ◽

Takashi Inoue ◽

Isamu Miyamoto

Keyword(s):

Laser Welding ◽

Co2 Laser ◽

Quantitative Characterization ◽

Welding Processes

Download Full-text

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Download Full-text