scholarly journals System-level studies of a cell-free transcription-translation platform for metabolic engineering

2017 ◽  
Author(s):  
Yong Y. Wu ◽  
Hirokazu Sato ◽  
Hongjun Huang ◽  
Stephanie J. Culler ◽  
Julia Khandurina ◽  
...  
Keyword(s):  
Design Space ◽  
Product Yield ◽  
System Level ◽  
Trial And Error ◽  
Strain Development ◽  
Design Build ◽  
A Cell ◽  
Metabolic Output

AbstractCurrent methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.

scholarly journals Characterizing and Prototyping Genetic Networks with Cell-Free Transcription-Translation Reactions

2015 ◽  
Author(s):  
Melissa K Takahashi ◽  
Clarmyra A. Hayes ◽  
James Chappell ◽  
Zachary Z. Sun ◽  
Richard M Murray ◽  
...  
Keyword(s):  
Gene Networks ◽  
Genetic Networks ◽  
Cellular Behavior ◽  
Network Function ◽  
Design Build ◽  
A Cell ◽  
In Vivo Characterization ◽  
Large Repertoire

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design-build-test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription-translation (TX-TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX-TL system that utilizes the native Escherichia coli TX-TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX-TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX-TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems.

Electric Field-Assisted Positioning of Neurons on Pt Microelectrode Arrays

2003 ◽  
Vol 773 ◽  
Author(s):  
Shalini Prasad ◽  
Mo Yang ◽  
Xuan Zhang ◽  
Yingchun Ni ◽  
Vladimir Parpura ◽  
...  
Keyword(s):  
Electric Field ◽  
Electrical Activity ◽  
Electric Fields ◽  
Microelectrode Array ◽  
Electrode Array ◽  
Sprague Dawley ◽  
A Cell ◽  
Neuron Patterning

AbstractCharacterization of electrical activity of individual neurons is the fundamental step in understanding the functioning of the nervous system. Single cell electrical activity at various stages of cell development is essential to accurately determine in in-vivo conditions the position of a cell based on the procured electrical activity. Understanding memory formation and development translates to changes in the electrical activity of individual neurons. Hence, there is an enormous need to develop novel ways for isolating and positioning individual neurons over single recording sites. To this end, we used a 3x3 multiple microelectrode array system to spatially arrange neurons by applying a gradient AC field. We characterized the electric field distribution inside our test platform by using two dimensiona l finite element modeling (FEM) and determined the location of neurons over the electrode array. Dielectrophoretic AC fields were utilized to separate the neurons from the glial cells and to position the neurons over the electrodes. The neurons were obtained from 0-2-day-old rat (Sprague-Dawley) pups. The technique of using electric fields to achieve single neuron patterning has implications in neural engineering, elucidating a new and simpler method to develop and study neuronal activity as compared to conventional microelectrode array techniques.

scholarly journals Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis inStreptomyces griseus DSM40695

2000 ◽  
Vol 44 (7) ◽  
pp. 1809-1817 ◽  
Author(s):  
Wyatt C. Smith ◽  
Longkuan Xiang ◽  
Ben Shen
Keyword(s):  
Polyketide Synthase ◽  
Isotope Labeling ◽  
Streptomyces Griseus ◽  
Open Reading Frames ◽  
A Cell ◽  
Cyclic Polyethers ◽  
The Difference ◽  
Reading Frames

ABSTRACT The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR andnonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of thenonS mutant by the expression of nonS intrans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of thenonS mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonSgene and fermentation of the nonS mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis inS. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

Disassembly of the Drosophila nuclear lamina in a homologous cell-free system

1995 ◽  
Vol 108 (5) ◽  
pp. 2027-2035 ◽  
Author(s):  
N. Maus ◽  
N. Stuurman ◽  
P.A. Fisher
Keyword(s):  
Nuclear Lamina ◽  
Meiotic Metaphase ◽  
Two Dimensional ◽  
Cell Free Extract ◽  
Free System ◽  
Nuclear Lamin ◽  
A Cell

Stage 14 Drosophila oocytes are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins. Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. A previously unidentified soluble lamin isoform is easily seen after in vitro disassembly. This isoform is detectable but present only in very small quantities in vivo and is apparently derived specifically from one of the two interphase lamin isoforms. Cell-free nuclear lamina disassembly is ATP-dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. This system will allow enzymological characterization of cell-free lamina disassembly as well as molecular analysis of specific Drosophila mutants.

scholarly journals A mutant allele of the Swi/Snf member BAF250a determines the pool size of fetal liver hemopoietic stem cell populations

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1678-1684 ◽  
Author(s):  
Jana Krosl ◽  
Aline Mamo ◽  
Jalila Chagraoui ◽  
Brian T. Wilhelm ◽  
Simon Girard ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Fetal Liver ◽  
Pool Size ◽  
Hemopoietic Stem Cell ◽  
Wild Type ◽  
A Cell ◽  
Dilution Experiments ◽  
Stem Cell Populations

Abstract It is believed that hemopoietic stem cells (HSC), which colonize the fetal liver (FL) rapidly, expand to establish a supply of HSCs adequate for maintenance of hemopoiesis throughout life. Accordingly, FL HSCs are actively cycling as opposed to their predominantly quiescent bone marrow counterparts, suggesting that the FL microenvironment provides unique signals that support HSC proliferation and self-renewal. We now report the generation and characterization of mice with a mutant allele of Baf250a lacking exons 2 and 3. Baf250aE2E3/E2E3 mice are viable until E19.5, but do not survive beyond birth. Most interestingly, FL HSC numbers are markedly higher in these mice than in control littermates, thus raising the possibility that Baf250a determines the HSC pool size in vivo. Limit dilution experiments indicate that the activity of Baf250aE2E3/E2E3 HSC is equivalent to that of the wild-type counterparts. The Baf250aE2E3/E2E3 FL-derived stroma, in contrast, exhibits a hemopoiesis-supporting potential superior to the developmentally matched controls. To our knowledge, this demonstration is the first that a mechanism operating in a cell nonautonomous manner canexpand the pool size of the fetal HSC populations.

scholarly journals Molecular Characterization of Serine-, Alanine-, and Proline-Rich Proteins of Trypanosoma cruzi and Their Possible Role in Host Cell Infection

2006 ◽  
Vol 74 (3) ◽  
pp. 1537-1546 ◽  
Author(s):  
Renata C. P. Baida ◽  
Márcia R. M. Santos ◽  
Mirian S. Carmo ◽  
Nobuko Yoshida ◽  
Danielle Ferreira ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Receptor Interaction ◽  
Cell Infection ◽  
Extracellular Medium ◽  
Metacyclic Trypomastigotes ◽  
A Cell

ABSTRACT We previously reported the isolation of a novel protein gene family, termed SAP (serine-, alanine-, and proline-rich protein), from Trypanosoma cruzi. Aided by the availability of the completed genome sequence of T. cruzi, we have now identified 39 full-length sequences of SAP, six pseudogenes and four partial genes. SAPs share a central domain of about 55 amino acids and can be divided into four groups based on their amino (N)- and carboxy (C)-terminal sequences. Some SAPs have conserved N- and C-terminal domains encoding a signal peptide and a glycosylphosphatidylinositol anchor addition site, respectively. Analysis of the expression of SAPs in metacyclic trypomastigotes by two-dimensional electrophoresis and immunoblotting revealed that they are likely to be posttranslationally modified in vivo. We have also demonstrated that some SAPs are shed into the extracellular medium. The recombinant SAP exhibited an adhesive capacity toward mammalian cells, where binding was dose dependent and saturable, indicating a possible ligand-receptor interaction. SAP triggered the host cell Ca2+ response required for parasite internalization. A cell invasion assay performed in the presence of SAP showed inhibition of internalization of the metacyclic forms of the CL strain. Taken together, these results show that SAP is involved in the invasion of mammalian cells by metacyclic trypomastigotes, and they confirm the hypothesis that infective trypomastigotes exploit an arsenal of surface glycoproteins and shed proteins to induce signaling events required for their internalization.

scholarly journals Amiodarone and Bepridil Inhibit Anthrax Toxin Entry into Host Cells

2007 ◽  
Vol 51 (7) ◽  
pp. 2403-2411 ◽  
Author(s):  
Ana M. Sanchez ◽  
Diane Thomas ◽  
Eugene J. Gillespie ◽  
Robert Damoiseaux ◽  
Joseph Rogers ◽  
...  
Keyword(s):  
Small Molecules ◽  
Lethal Toxin ◽  
Host Cells ◽  
Anthrax Lethal Toxin ◽  
Raw 264.7 Macrophages ◽  
Endosomal Acidification ◽  
A Cell

ABSTRACT Anthrax lethal toxin is one of the fundamental components believed to be responsible for the virulence of Bacillus anthracis. In order to find novel compounds with anti-lethal toxin properties, we used a cell-based assay to screen a collection of approximately 500 small molecules. Nineteen compounds that blocked lethal toxin-mediated killing of RAW 264.7 macrophages were identified, and we report here on the characterization of the two most potent antitoxic compounds, amiodarone and bepridil. These drugs are used to treat cardiac arrhythmia or angina in humans at doses similar to those that provide protection against lethal toxin in vitro. Our results support a model whereby the antitoxic properties of both drugs result from their ability to block endosomal acidification, thereby blocking toxin entry. Amiodarone was tested in vivo and found to significantly increase survival of lethal toxin-challenged Fischer rats.

Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody

2016 ◽  
Vol 58 (8-9) ◽  
pp. 585-594 ◽  
Author(s):  
Galina Gulis ◽  
Izabel Cristina Rodrigues Silva ◽  
Herdson Renney Sousa ◽  
Isabel Garcia Sousa ◽  
Maryani Andressa Gomes Bezerra ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Dna Detection ◽  
Detection Probe ◽  
A Cell ◽  
Z Dna

scholarly journals Cell-free andin vivocharacterization of Lux, Las, and Rpa quorum activation systems inE. coli

2017 ◽  
Author(s):  
Andrew D. Halleran ◽  
Richard M. Murray
Keyword(s):  
Quorum Sensing ◽  
Design Space ◽  
Group Decision ◽  
Microbial Consortia ◽  
Test Bed ◽  
Free System ◽  
Sensing Systems ◽  
Signal Crosstalk

AbstractSynthetic biologists have turned towards quorum systems as a path for building sophisticated microbial consortia that exhibit group decision making. Currently, however, even the most complex consortium circuits rely on only one or two quorum sensing systems, greatly restricting the available design space. High-throughput characterization of available quorum sensing systems is useful for finding compatible sets of systems that are suitable for a defined circuit architecture. Recently, cell–free systems have gained popularity as a test-bed for rapid prototyping of genetic circuitry.We take advantage of the transcription-translation cell-free system to characterize three commonly used Lux-type quorum activators, Lux, Las, and Rpa. We then compare the cell-free characterization to results obtainedin vivo.We find significant genetic crosstalk in both the Las and Rpa systems and substantial signal crosstalk in Lux activation. We show that cell-free characterization predicts crosstalk observedin vivo.

scholarly journals Characterization of cimetidine transport in LLCPK1 cells.

1994 ◽  
Vol 5 (1) ◽  
pp. 75-84
Author(s):  
R Bendayan ◽  
B Lo ◽  
M Silverman
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Potent Inhibitor ◽  
Organic Base ◽  
Temperature Dependent ◽  
Exchange System ◽  
Excess Concentration ◽  
A Cell

In this study, cimetidine uptake and its regulation by LLCPK1 monolayers were investigated. Uptake was temperature dependent with kinetic and specificity characteristics typical of a carrier-mediated mechanism. With cimetidine uptake in the presence of an excess concentration of the potent inhibitor quinidine as a measure of nonspecific transport, the estimated kinetic parameters for cimetidine uptake at 37 degrees C under steady-state conditions are Km = 32.3 +/- 6.4 microM and Vmax = 20.2 +/- 2.1 pmol/mg per minute. Amiloride, quinidine, and quinine inhibited cimetidine uptake, whereas N1-methylnicotinamide, tetraethylammonium, and guanidine did not. The uptake of cimetidine was increased in the presence of a cell-->lumen H+ gradient, consistent with the behavior of a cimetidine-H+ antiport system. Furthermore, the activity of both the Na(+)-H+ exchanger and H(+)-ATPase acted to dissipate the cell-->lumen H+ gradient, thereby decreasing net cimetidine transport. These results suggest that there is a cimetidine-H+ exchange system in LLCPK1 cells and that the net secretion of organic base in vivo may be regulated by luminal acidification mechanisms.

Sign in / Sign up

Export Citation Format

Share Document