scholarly journals Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation

Bioengineered ◽  
2017 ◽  
Vol 9 (1) ◽  
pp. 90-97 ◽  
Author(s):  
Conner C. Earl ◽  
Mark T. Smith ◽  
Richard A. Lease ◽  
Bradley C. Bundy
Keyword(s):  
Low Cost ◽  
Protein Translation ◽  
Rnase Inhibitor ◽  
Free Protein ◽  
Polyvinylsulfonic Acid ◽  
Rna Preservation

scholarly journals Instrument-Free Protein Microarray Fabrication for Accurate Affinity Measurements

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 158
Author(s):  
Iris Celebi ◽  
Matthew T. Geib ◽  
Elisa Chiodi ◽  
Nese Lortlar Ünlü ◽  
Fulya Ekiz Kanik ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Low Cost ◽  
Protein Microarray ◽  
Active Surface ◽  
Protein Microarrays ◽  
Free Protein ◽  
Microarray Fabrication ◽  
Antigen Antibody ◽  
Imaging Sensor ◽  
Protein Assays

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have a higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in a 24-multiplexed chip format with less than 5% measurement error.

Instrument-Free Protein Microarray Fabrication for Accurate Affinity Measurements

2020 ◽  
Author(s):  
Iris Celebi ◽  
Matthew T. Geib ◽  
Elisa Chiodi ◽  
Nese Lortlar Ünlü ◽  
Fulya Ekiz Kanik ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Low Cost ◽  
Protein Microarray ◽  
Active Surface ◽  
Protein Microarrays ◽  
Free Protein ◽  
Microarray Fabrication ◽  
Antigen Antibody ◽  
Imaging Sensor ◽  
Protein Assays

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-Lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-Lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging of 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in 24-multiplexed chip format with less than 5% measurement error.

scholarly journals Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths

2017 ◽  
Author(s):  
Sabrina Galiñanes Reyes ◽  
Yutetsu Kuruma ◽  
Soichiro Tsuda
Keyword(s):  
Protein Expression ◽  
Systems Approach ◽  
Protein Translation ◽  
Limiting Factors ◽  
T7 Promoter ◽  
Free System ◽  
Free Protein ◽  
Pure System ◽  
E Coli ◽  
Promoter Library

AbstractCell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to express proteins at different transcription rates in a reconstituted and E. coli extract-based cell-free systems. We find that the maximum expression level and the rate of protein synthesis as responses to the transcription rate change are different in the two cell-free systems, suggesting they are driven by different expression dynamics. We confirm this by constructing a simple mathematical model for each cell-free system, which well reproduce the experimental results and also identify different limiting factors for better protein expression in the two cell-free systems. In particular, they revealed there is a negative feedback effect in the mRNA-protein translation by the PURE system and also identified different limiting factors for better protein expression in the two cell-free systems.

Instrument-Free Protein Microarray Fabrication for Accurate Affinity Measurements

2020 ◽  
Author(s):  
Iris Celebi ◽  
Matthew T. Geib ◽  
Elisa Chiodi ◽  
Nese Lortlar Ünlü ◽  
Fulya Ekiz Kanik ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Low Cost ◽  
Protein Microarray ◽  
Active Surface ◽  
Protein Microarrays ◽  
Free Protein ◽  
Microarray Fabrication ◽  
Antigen Antibody ◽  
Imaging Sensor ◽  
Protein Assays

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-Lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-Lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging of 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in 24-multiplexed chip format with less than 5% measurement error.

scholarly journals Decentralizing cell-free RNA sensing with the use of low-cost cell extracts

2021 ◽  
Author(s):  
Anibal Arce ◽  
Fernando Guzman ◽  
Chiara Gandini ◽  
Juan Puig ◽  
Tamara Matute ◽  
...  
Keyword(s):  
Protein Expression ◽  
Low Cost ◽  
Cold Chain ◽  
Expression Systems ◽  
Free Protein ◽  
Cell Extracts ◽  
Freeze Dried ◽  
Rna Sensors ◽  
Pure Cell ◽  
Pcr Products

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Here, we describe the implementation of RNA toehold switch-based sensors using E. coli cell lysate-based cell-free protein expression systems, which can be produced locally and reduce the cost of sensors by two orders of magnitude. We then demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize use of these lysates with a CRISPRi strategy to enhance the stability of linear DNAs, enabling the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toeholds sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

Decomposition of the metastable beta titanium alloy Ti-15-3

1983 ◽  
Vol 41 ◽  
pp. 264-265
Author(s):  
Y. L. Chen ◽  
S. Fujlshiro
Keyword(s):  
Low Cost ◽  
High Strength ◽  
Alpha Phase ◽  
Thick Sheet ◽  
Omega Phase ◽  
Beta Titanium Alloy ◽  
Beta Titanium ◽  
Beta Matrix ◽  
Metastable Beta ◽  
Very High

Metastable beta titanium alloys have been known to have numerous advantages such as cold formability, high strength, good fracture resistance, deep hardenability, and cost effectiveness. Very high strength is obtainable by precipitation of the hexagonal alpha phase in a bcc beta matrix in these alloys. Precipitation hardening in the metastable beta alloys may also result from the formation of transition phases such as omega phase. Ti-15-3 (Ti-15V- 3Cr-3Al-3Sn) has been developed recently by TIMET and USAF for low cost sheet metal applications. The purpose of the present study was to examine the aging characteristics in this alloy.The composition of the as-received material is: 14.7 V, 3.14 Cr, 3.05 Al, 2.26 Sn, and 0.145 Fe. The beta transus temperature as determined by optical metallographic method was about 770°C. Specimen coupons were prepared from a mill-annealed 1.2 mm thick sheet, and solution treated at 827°C for 2 hr in argon, then water quenched. Aging was also done in argon at temperatures ranging from 316 to 616°C for various times.

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