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<h1> | <h1>Abstract</h1> | ||
Biological systems primarily use proteins to sense and respond to other molecules. We sought to engineer a protein-based platform as the basis of a biologically emulative biosensor. This biosensor platform is especially powerful if both the analyte sensitivity and transduction method can be straightforwardly customized to respond to important targets. Here we show our attempts to demonstrate the modularity of a pre-existing protein biosensor through such customization. The calcium-binding protein calmodulin has already been engineered to exhibit enzymatic switching in response to peptide binding through fusion to a split TEM1 ß-lactamase. To extend this platform, we first evolved the calmodulin-ß-lactamase fusion (BlaCaM) to exhibit sensitivity to a previously inactive peptide, Staphylococcus aureus ∂-toxin, through random mutagenesis of the calmodulin portion of BlaCaM, followed by screening the purified library for ß-lactamase activity. Second, we altered the transduction domain by exchanging the output domain of BlaCaM with a split Gaussia luciferase, replacing the existing output signal, ß-lactam degradation, with luminescence. Gaussia luciferase was fragmented according to a known split site and different linkers were designed for variable length and rigidity to maximize switching activity. Our attempts demonstrate the possibility of a modular, calmodulin-based biosensor that can be easily designed with varying input specificity and output modes. | |||
Revision as of 19:00, 18 October 2013
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HARVARD
BIODESIGN
BIOMOD 2013
-
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard"><li>HOME</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/introduction"><li>INTRODUCTION</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/design"><li>DESIGN</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/methods"><li>METHODS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/protocols"><li>PROTOCOLS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/team"><li>TEAM</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/lab"><li>LAB NOTEBOOK</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/results"><li>RESULTS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/references"><li>REFERENCES</li></a></html>
Abstract
Biological systems primarily use proteins to sense and respond to other molecules. We sought to engineer a protein-based platform as the basis of a biologically emulative biosensor. This biosensor platform is especially powerful if both the analyte sensitivity and transduction method can be straightforwardly customized to respond to important targets. Here we show our attempts to demonstrate the modularity of a pre-existing protein biosensor through such customization. The calcium-binding protein calmodulin has already been engineered to exhibit enzymatic switching in response to peptide binding through fusion to a split TEM1 ß-lactamase. To extend this platform, we first evolved the calmodulin-ß-lactamase fusion (BlaCaM) to exhibit sensitivity to a previously inactive peptide, Staphylococcus aureus ∂-toxin, through random mutagenesis of the calmodulin portion of BlaCaM, followed by screening the purified library for ß-lactamase activity. Second, we altered the transduction domain by exchanging the output domain of BlaCaM with a split Gaussia luciferase, replacing the existing output signal, ß-lactam degradation, with luminescence. Gaussia luciferase was fragmented according to a known split site and different linkers were designed for variable length and rigidity to maximize switching activity. Our attempts demonstrate the possibility of a modular, calmodulin-based biosensor that can be easily designed with varying input specificity and output modes.
