Application of mRNA Arrays for the Production of mCherry Reporter-Protein Arrays for Quantitative Gene Expression Analysis

Abstract

The development of programmable regulators that precisely and predictably control gene expression is a major goal of synthetic biology. Consequently, rapid high-throughput biochemical methods capable of quantitatively analyzing all components of gene expression would be of value in the characterization and optimization of regulator performance. In this study we demonstrate a novel application of RNA arrays, involving the production of reporterprotein arrays, to gene expression analysis. This method enables simultaneous quantification of both the transcription and posttranscription/translation components of gene expression, and it also allows the assessment of the orthogonality of multiple regulators. We use our method to directly compare the performance of a series of previously characterized synthetic post-transcriptional riboregulators, thus demonstrating its utility in the development of synthetic regulatory modules and evaluation of gene expression regulation in general.

Rapid Fabrication of Protein Microarrays via Autogeneration and on-Chip Purification of Biotinylated Probes

“…3−5 The strongest noncovalent linkage known in nature, the binding of biotin to avidin or streptavidin, is extremely tight and specific under a wide range of conditions, making this binding an excellent "glue" for "sticking" protein probes onto a microarray surface. 4,5 Consequently, the effective production of biotinylated protein probes is important as a prerequisite for anchoring the affinity protein through the biotin−streptavidin linkage.…”

“…As a powerful tool used for many purposes, including applications in clinical diagnosis, drug and vaccine discovery, and laboratory research, miniaturized and parallel protein microarrays can screen numerous proteins simultaneously in an automated fashion with low consumption of sample. The availability of a protein microarray is mainly determined by both the accessibility of a defined set of probes and the methodology for their generation and immobilization with well-preserved functionality. , Compared with probes that use direct adsorption and covalent coupling, noncovalent capturing via their specific ligands is regarded as a more effective immobilization method, which can provide better immobilization and less functional repression of the protein probes. − The strongest noncovalent linkage known in nature, the binding of biotin to avidin or streptavidin, is extremely tight and specific under a wide range of conditions, making this binding an excellent “glue” for “sticking” protein probes onto a microarray surface. , Consequently, the effective production of biotinylated protein probes is important as a prerequisite for anchoring the affinity protein through the biotin–streptavidin linkage.…”

Reprogramming Gene Expression by Targeting RNA-Based Interactions: A Novel Pipeline Utilizing RNA Array Technology

“…We recently developed an innovative “sandwich” method for the production of functional-RNA arrays, involving in vitro transcription of a DNA template array and in situ RNA capture on a facing surface . These RNA arrays can be used as a platform for investigating multiple RNA-based interactions, including sRNA–mRNA interactions, in parallel. ,, Consequently, we decided to use this technology to generate a Qrr sRNA array, to be used as the basis for a Qrr sRNA– hapR mRNA interaction assay (Figure ).…”

How to copy and paste DNA microarrays

“…We think that the presented DNA copies only represent the first step of microarray copying. Since the DNA molecules of the original DNA microarray are immobilised inside the cavities of the master cavity chip, RNA 21,22 or protein 23–26 copies could also be realised. This can be done by changing the copy surface and biochemical mix.…”