A novel method for rapid and sensitive metagenomic activity screening
| Autor: | Dominic W. S. Wong, Meiling Shang, Victor J. Chan, Hans Liao |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
030103 biophysics Clinical Biochemistry Computational biology Activity screening law.invention Agar plate Novel gene 03 medical and health sciences law Biochemistry Genetics and Molecular Biology Functional screening Split-pool sub-library screening lcsh:Science ComputingMethodologies_COMPUTERGRAPHICS Cloning Substrate gel microtiter assay plate Chemistry Petri dish Substrate (chemistry) Isolation (microbiology) Medical Laboratory Technology 030104 developmental biology Metagenomics lcsh:Q Metagenomic library Gene Discovery |
| Zdroj: | MethodsX, Vol 5, Iss, Pp 669-675 (2018) MethodsX |
| ISSN: | 2215-0161 |
| DOI: | 10.1016/j.mex.2018.06.011 |
| Popis: | Graphical abstract Direct cloning of metagenomes has proven to be a powerful tool for the exploration of the diverse sequence space of a microbial community leading to gene discovery and biocatalyst development. The key to such approach is the development of rapid, sensitive, and reliable functional screening of libraries. The majority of library screen have relied on the use of agar plates in petri dishes incorporating the target enzyme substrate for activity detection of positive clones (Iqbal et al. [1], Knietsch et al. [2], Popovic et al. [3]). In this article, a novel method is described consisting of: (1) formulation and application of substrate gel microtiter assay plates, (2) screening of libraries of clones in split pools in the wells of the assay plate, and (3) progressive enrichment and isolation of individual positive clones. The method has been successfully used in the rapid discovery of novel genes and enzymes from rumen microbial metagenome with high efficacy. • Novel substrate gel assay plates for activity screening with localized and intensified signals. • Rapid and complete screening of library clones in split pools. • Progressive enrichment scheme as a refining step for isolating target gene. |
| Databáze: | OpenAIRE |
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