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A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors.
BMC Biotechnol. 2011 Nov 10; 11:105.BB

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%).

Authors+Show Affiliations

Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22074568

Citation

Ibraheem, Andreas, et al. "A Bacteria Colony-based Screen for Optimal Linker Combinations in Genetically Encoded Biosensors." BMC Biotechnology, vol. 11, 2011, p. 105.
Ibraheem A, Yap H, Ding Y, et al. A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors. BMC Biotechnol. 2011;11:105.
Ibraheem, A., Yap, H., Ding, Y., & Campbell, R. E. (2011). A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors. BMC Biotechnology, 11, 105. https://doi.org/10.1186/1472-6750-11-105
Ibraheem A, et al. A Bacteria Colony-based Screen for Optimal Linker Combinations in Genetically Encoded Biosensors. BMC Biotechnol. 2011 Nov 10;11:105. PubMed PMID: 22074568.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors. AU - Ibraheem,Andreas, AU - Yap,Hongkin, AU - Ding,Yidan, AU - Campbell,Robert E, Y1 - 2011/11/10/ PY - 2011/09/08/received PY - 2011/11/10/accepted PY - 2011/11/15/entrez PY - 2011/11/15/pubmed PY - 2012/2/10/medline SP - 105 EP - 105 JF - BMC biotechnology JO - BMC Biotechnol. VL - 11 N2 - 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%). SN - 1472-6750 UR - https://www.unboundmedicine.com/medline/citation/22074568/A_bacteria_colony_based_screen_for_optimal_linker_combinations_in_genetically_encoded_biosensors_ L2 - https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-11-105 DB - PRIME DP - Unbound Medicine ER -