Please use this identifier to cite or link to this item:
|Title:||A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors|
|Publisher:||BioMed Central Ltd.|
|Source:||BMC biotechnology, 2011, v. 11, 105, p. 1-13 How to cite?|
|Abstract:||Background: Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.|
Results: To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.
Conclusion: Application of this library screening methodology led to the identification of an optimized H3K27- trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%).
|Rights:||© 2011 Ibraheem et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.|
|Appears in Collections:||Journal/Magazine Article|
Show full item record
Files in This Item:
|Ibraheem_Bacteria_colony-based.pdf||2.84 MB||Adobe PDF||View/Open|
Citations as of Jun 2, 2016
WEB OF SCIENCETM
Citations as of Sep 24, 2016
Checked on Oct 16, 2016
Checked on Oct 16, 2016
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.