On the Design of Low-Cost Fluorescent Protein Biosensors

2009 ◽  
pp. 99-123 ◽  
Author(s):  
Leah Tolosa
Keyword(s):  
Fluorescent Protein ◽  
Low Cost

scholarly journals rClone Red facilitates bacterial gene expression research by undergraduates in the teaching laboratory

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
A Malcolm Campbell ◽  
Todd T Eckdahl
Keyword(s):  
Binding Sites ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Undergraduate Teaching ◽  
Original Research ◽  
Rrna Gene ◽  
Translation Efficiency ◽  
Red Fluorescent Protein ◽  
Translational Initiation ◽  
Bacterial Gene

Abstract rClone Red is a low-cost and student-friendly research tool that has been used successfully in undergraduate teaching laboratories. It enables students to perform original research within the financial and time constraints of a typical undergraduate environment. Students can strengthen their understanding of the initiation of bacterial translation by cloning ribosomal binding sites of their own design and using a red fluorescent protein reporter to measure translation efficiency. Online microbial genome sequences and the mFold website enable students to explore homologous rRNA gene sequences and RNA folding, respectively. In this report, we described how students in a genetics course who were given the opportunity to use rClone Red demonstrated significant learning gains on 16 of 20 concepts, and made original discoveries about the function of ribosome binding sites. By combining the highly successful cloning method of golden gate assembly with the dual reporter proteins of green fluorescent protein and red fluorescent protein, rClone Red enables novice undergraduates to make new discoveries about the mechanisms of translational initiation, while learning the core concepts of genetic information flow in bacteria.

scholarly journals Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom

2020 ◽  
Vol 1 (2) ◽  
Author(s):  
John W. Rupel ◽  
Sophia M. Sdao ◽  
Kadina E. Johnston ◽  
Ethan T. Nethery ◽  
Kaitlyn A. Gabardi ◽  
...  
Keyword(s):  
Undergraduate Students ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Low Cost ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Light Sources ◽  
Effective Manner ◽  
Primary Mouse

ABSTRACT Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.

scholarly journals Bioluminescent Ratiometric Indicator for Analysis of Water Hardness in Household Water

Sensors ◽  
2020 ◽  
Vol 20 (11) ◽  
pp. 3164
Author(s):  
Md Nadim Hossain ◽  
Ryuichi Ishida ◽  
Mitsuru Hattori ◽  
Tomoki Matsuda ◽  
Takeharu Nagai
Keyword(s):  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Negative Impact ◽  
Low Cost ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Water Hardness ◽  
Hard Water ◽  
Household Water

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.

scholarly journals CCD Based Detector for Detection of Abrin Toxin Activity

Toxins ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 120
Author(s):  
Reuven Rasooly ◽  
Paula Do ◽  
Bradley Hernlem
Keyword(s):  
Hela Cells ◽  
Thermal Inactivation ◽  
Fluorescent Protein ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Low Cost ◽  
Vero Cells ◽  
Multiple Organ ◽  
Biologically Active ◽  
Organ Systems

Abrin is a highly potent and naturally occurring toxin produced in the seeds of Abrus precatorius (Rosary Pea) and is of concern as a potential bioterrorism weapon. There are many rapid and specific assay methods to detect this toxic plant protein, but few are based on detection of toxin activity, critical to discern biologically active toxin that disables ribosomes and thereby inhibits protein synthesis, producing cytotoxic effects in multiple organ systems, from degraded or inactivated toxin which is not a threat. A simple and low-cost CCD detector system was evaluated with colorimetric and fluorometric cell-based assays for abrin activity; in the first instance measuring the abrin suppression of mitochondrial dehydrogenase in Vero cells by the MTT-formazan method and in the second instance measuring the abrin suppression of green fluorescent protein (GFP) expression in transduced Vero and HeLa cells. The limit of detection using the colorimetric assay was 10 pg/mL which was comparable to the fluorometric assay using HeLa cells. However, with GFP transduced Vero cells a hundred-fold improvement in sensitivity was achieved. Results were comparable to those using a more expensive commercial plate reader. Thermal inactivation of abrin was studied in PBS and in milk using the GFP-Vero cell assay. Inactivation at 100 °C for 5 min in both media was complete only at the lowest concentration studied (0.1 ng/mL) while treatment at 63 °C for 30 min was effective in PBS but not milk.

scholarly journals Inducible and tunable gene expression systems for Pseudomonas putida KT2440

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chandran Sathesh-Prabu ◽  
Rameshwar Tiwari ◽  
Doyun Kim ◽  
Sung Kuk Lee
Keyword(s):  
Pseudomonas Putida ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Inducible Promoter ◽  
Expression Systems ◽  
Reporter Protein ◽  
Pseudomonas Putida Kt2440 ◽  
Wide Range ◽  
Green Fluorescent

AbstractInducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/PhpdH was also efficiently induced by LA. LvaR/PlvaA and XutR/PxutA systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/Pzwf1 showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.

scholarly journals A versatile and customizable low-cost 3D-printed open standard for microscopic imaging

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Benedict Diederich ◽  
René Lachmann ◽  
Swen Carlstedt ◽  
Barbora Marsikova ◽  
Haoran Wang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Low Cost ◽  
Optical Resolution ◽  
Light Sheet ◽  
Transgenic Zebrafish ◽  
Development Cycle ◽  
Open Standard ◽  
Resolution Level ◽  
3D Printed ◽  
Green Fluorescent

AbstractModern microscopes used for biological imaging often present themselves as black boxes whose precise operating principle remains unknown, and whose optical resolution and price seem to be in inverse proportion to each other. With UC2 (You. See. Too.) we present a low-cost, 3D-printed, open-source, modular microscopy toolbox and demonstrate its versatility by realizing a complete microscope development cycle from concept to experimental phase. The self-contained incubator-enclosed brightfield microscope monitors monocyte to macrophage cell differentiation for seven days at cellular resolution level (e.g. 2 μm). Furthermore, by including very few additional components, the geometry is transferred into a 400 Euro light sheet fluorescence microscope for volumetric observations of a transgenic Zebrafish expressing green fluorescent protein (GFP). With this, we aim to establish an open standard in optics to facilitate interfacing with various complementary platforms. By making the content and comprehensive documentation publicly available, the systems presented here lend themselves to easy and straightforward replications, modifications, and extensions.

scholarly journals Promoter engineering for microbial bio-alkane gas production

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Duangthip Trisrivirat ◽  
John M X Hughes ◽  
Robin Hoeven ◽  
Matthew Faulkner ◽  
Helen Toogood ◽  
...  
Keyword(s):  
Lower Cost ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Gas Production ◽  
Low Carbon ◽  
E Coli ◽  
Promoter Library ◽  
Production Strategy ◽  
Inducible Systems ◽  
The Cost

Abstract Successful industrial biotechnological solutions to biofuels and other chemicals production rely on effective competition with existing lower-cost natural sources and synthetic chemistry approaches enabled by adopting low-cost bioreactors and processes. This is achievable by mobilizing Halomonas as a next generation industrial chassis, which can be cultivated under non-sterile conditions. To increase the cost effectiveness of an existing sustainable low carbon bio-propane production strategy, we designed and screened a constitutive promoter library based on the known strong porin promoter from Halomonas. Comparative studies were performed between Escherichia coli and Halomonas using the reporter gene red fluorescent protein (RFP). Later studies with a fatty acid photodecarboxylase-RFP fusion protein demonstrated tuneable propane production in Halomonas and E. coli, with an ∼8-fold improvement in yield over comparable isopropyl-β-D-thiogalactoside-inducible systems. This novel set of promoters is a useful addition to the synthetic biology toolbox for future engineering of Halomonas to make chemicals and fuels.

scholarly journals Construction of a mApple-D6A3-mediated biosensor for detection of heavy metal ions

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yangyang Ji ◽  
Feifei Guan ◽  
Xin Zhou ◽  
Xiaoqing Liu ◽  
Ningfeng Wu ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Metal Ions ◽  
Fluorescence Intensity ◽  
Heavy Metal Ions ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Detection Methods ◽  
Titration Calorimetry ◽  
Efficient Detection

AbstractPollution of heavy metals in agricultural environments is a growing problem to the health of the world’s human population. Green, low-cost, and efficient detection methods can help control such pollution. In this study, a protein biosensor, mApple-D6A3, was built from rice-derived Cd2+-binding protein D6A3 fused with the red fluorescent protein mApple at the N-terminus to detect the contents of heavy metals. Fluorescence intensity of mApple fused with D6A3 indicated the biosensor’s sensitivity to metal ions and its intensity was more stable under alkaline conditions. mApple-D6A3 was most sensitive to Cu2+, then Ni2+, then Cd2+. Isothermal titration calorimetry experiments demonstrated that mApple-D6A3 successfully bound to each of these three metal ions, and its ability to bind the ions was, from strongest to weakest, Cu2+  > Cd2+  > Ni2+. There were strong linear relationships between the fluorescence intensity of mApple-D6A3 and concentrations of Cd2+ (0–100 μM), Cu2+ (0–60 μM) and Ni2+ (0–120 μM), and their respective R2 values were 0.994, 0.973 and 0.973. When mApple-D6A3 was applied to detect concentrations of heavy metal ions in water (0–0.1 mM) or culture medium (0–1 mM), its accuracy for detection attained more than 80%. This study demonstrates the potential of this biosensor as a tool for detection of heavy metal ions.

scholarly journals Green Fluorescent Protein Reporter Microplate Assay for High-Throughput Screening of Compounds againstMycobacterium tuberculosis

1998 ◽  
Vol 42 (2) ◽  
pp. 344-347 ◽  
Author(s):  
L. A. Collins ◽  
M. N. Torrero ◽  
S. G. Franzblau
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Firefly Luciferase ◽  
Drug Susceptibility ◽  
Luciferase Reporter ◽  
Microplate Assay ◽  
Green Fluorescent

ABSTRACT An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H37Ra and M. tuberculosisH37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.

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