scholarly journals High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui Jiang ◽  
Donglei Jiang ◽  
Jingdong Shao ◽  
Xiulan Sun ◽  
Jiasheng Wang
Keyword(s):  
High Throughput ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Optical Biosensor ◽  
Bacterial Lipopolysaccharide ◽  
Red Fluorescent Protein ◽  
Reporter System ◽  
Fluorescent Protein Reporter

scholarly journals Sorting out antibiotics' mechanisms of action: a double fluorescent protein reporter for high throughput screening of ribosome and DNA biosynthesis inhibitors

2016 ◽  
pp. AAC.02117-16 ◽  
Author(s):  
Ilya A. Osterman ◽  
Ekaterina S. Komarova ◽  
Dmitry I. Shiryaev ◽  
Ilya A. Korniltsev ◽  
Irina M. Khven ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cost Effective ◽  
High Signal ◽  
Red Fluorescent Protein ◽  
Reporter System ◽  
Ribosome Stalling

In order to accelerate drug discovery, a simple, reliable and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double reporter system for not only antimicrobial activity detection, but also for simultaneous sorting of potential antimicrobials into those that cause ribosome stalling, and others that induce SOS response due to DNA damage. In this reporter system the red fluorescent protein generfpwas placed under the control of the SOS-induciblesulApromoter. The far-red fluorescent protein genekatushka2Swas inserted downstream the tryptophan attenuator where two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator, to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to any ribosome stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need in enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.

Porcine OCT4 Reporter System can Monitor Species-Specific Pluripotency During Somatic Cell Reprogramming

2020 ◽  
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Mingyun Lee ◽  
Dong-Kyung Lee ◽  
Chang-Kyu Lee
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Upstream Region ◽  
Reporter System ◽  
System L ◽  
Reporter Systems ◽  
Induced Pluripotent ◽  
Species Specific ◽  
Fluorescent Protein Reporter

Abstract l Background: The present study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in FGF- or LIF-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. l Results: Porcine embryonic fibroblasts were coinfected with the pOCT4-∆PE-eGFP (DE-GFP) and pOCT4-∆DE-DsRed2 (PE-RFP) vectors, and GFP and RFP expression was verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated in different expression patterns simultaneously as the changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). l Conclusions: Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems serve as live naïve/primed pluripotency indicators for porcine induced pluripotent cell establishment. This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development and embryonic stem cells.

scholarly journals A Reporter System for Reverse Transfection Cell Arrays

2003 ◽  
Vol 8 (6) ◽  
pp. 620-623 ◽  
Author(s):  
Brian L. Webb ◽  
Begoña Díaz ◽  
G. Steven Martin ◽  
Fang Lai
Keyword(s):  
Map Kinase ◽  
High Throughput ◽  
Gene Function ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Signaling Proteins ◽  
Kinase Signaling ◽  
Reporter System ◽  
Map Kinase Signaling ◽  
Reverse Transfection

The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conven tional gene discovery approaches in the pharmaceutical industry. High-throughput approaches for studying gene function in vivo are greatly needed. One potential answer to this challenge is reverse transfection, a high-throughput gene expression method for examining the function of hundreds to thousands of genes in parallel. One limitation of reverse transfection tech nology is the need for posttransfection processing of the arrays to analyze the activity of the expressed proteins. The authors have investigated the use of a reporter construct cotransfected with other genes of interest to monitor and screen gene function on reverse transfection microarrays. They developed a serum response element (SRE) reporter linked to the green fluorescent protein (GFP) that is cotransfected with target genes on reverse transfection arrays for monitoring mitogen-activated protein (MAP) kinase signaling by multiple targets in parallel. The authors show that this reporter system is able to detect inhibition of upstream MAP kinase signaling proteins by the MEK inhibitor U0126. The ability to monitor the activity of multiple signaling proteins in a multiwell format suggests the utility of reverse transfection reporter arrays for high-throughput screening applications.

scholarly journals Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

2003 ◽  
Vol 47 (12) ◽  
pp. 3682-3687 ◽  
Author(s):  
Chartchai Changsen ◽  
Scott G. Franzblau ◽  
Prasit Palittapongarnpim
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Enhanced Fluorescence ◽  
Signal To Noise ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

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