Biomod/2013/Harvard/design
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HARVARD
BIODESIGN
BIOMOD 2013
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<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>
Design
Biosensors
A quick and accurate detection of bioagents such as toxins and clinically significant biomarkers plays an essential role in biotechnology, medicine, agriculture, and even in military. One approach for detecting bioagents is the use of biosensors. Biosensors are biologically derived chemical sensing device that recognizes a presence of a certain molecule and outputs a measurable signal in response. It is composed of two parts: the bio-element that recognizes a specific analyte, or bioagent, and the transducer that converts the recognition into a readily detectable output signal.
Modular Platform for Allosteric Switches
Allosteric proteins, proteins that change shape when bound to another molecule, serve as a powerful platform for biosensors. With careful engineering, the ability to change conformation can be harnessed to switch on and off a signal, forming a simple biosensor. By attaching two halves of a transducing enzyme to an allosteric framework, the enzyme's activity can be regulated by the conformational changes of the protein it is bound to, and thus by the concentration of the protein's substrate. Techniques make it possible to vary both the output protein and the substrate of the allosteric framework, in theory allowing modular construction of a biosensor. This goal of modularity, summarized by Meister & Joshi, 2013 in the quote below, is the focus of Harvard BioDesign 2013's project.
"Our long-term goal is to develop a modular platform for peptide biosensing in which several input and output domains can be independently optimized using a combination of directed evolution and rational design methods, then combined to create a sensor with the desired input-output functions."
Input
The goal of the input team is to improve the binding and switching activity of the BlaCaM protein with respect to a previously non-functional analyte. A successful evolution would demonstrate the ability of the BlaCaM switch to sense different molecules, highlighting its potential as a biosensor component. BlaCaM is a fusion of two proteins, a calmodulin center with two halves of β-lactamase attached to the N- and C-termini. Calmodulin displays large conformational changes when it binds to both calcium and varying peptides.
Directed evolution was performed by screening individual purified members of libraries for activity toward a previously inactive peptide, Staphylococcus aureusδ-toxin. The Calmodulin portion of the BlaCaM fusion gene was mutagenized randomly and cells expressing those mutants were grown in 96-well plates. The expressed BlaCaM library members were purified via a 6xHis tag and screened for δ-toxin-activated β-lactamase activity in vitro. Members showing increased δ-toxin activity relative to a negative and positive control were re-screened to verify, and then combined and mutagenized again to continue a second generation of the evolution.
The main method driving our directed evolution is bacterial display. In bacterial display, the mutated BlaCaM proteins are displayed upon the surface of bacterial, allowing the proteins to interact with compounds outside of the bacterial. To move the proteins to the outside of the cell, their genes are cloned into bacteria fused to a transporter protein that facilitates transport from the cytoplasm to the surface of the tell. These displaying bacteria are then washed over a media displaying anchored versions of our analyte. Displayed proteins that have been successfully mutated to bind to the analyte will remain fixed to the media, while unsuccessful mutations will be washed away. The bound bacteria are then concentrated, isolated, and analyzed to determine the sequence of the evolved proteins displayed upon their surfaces.

Output
The output team's goal is to create a protein from Guassia princeps-derived luciferase (GLuc) and calmodulin (CaM) that will luminesce upon a conformational change of CaM. To create this protein, the output team will have to split GLuc and then fuse a part of it to each arm of the CaM molecule using a linker (see figure below).
The protein will need to be designed such that when CaM changes conformation due to the presence of calcium and the target peptide, the two halves of GLuc will come together and output chemiluminescence that can be detected. We will have to optimize the cut site of GLuc (building off the work done by Kim et al, 2009), and optimize the linkers so that the two halves of GLuc will come together when and only when CaM changes confirmation due to the presence of the target peptide.
In order for our protein to work as an effective biosensor, it must be reversible so that the switch can "turn off" and cease to luminesce when taken out of contact with the target peptide. Therefore the GLuc halves must be designed so that they can come apart and return to their original locations when the target peptide is no longer present. Enabling this reversibility will be one of the primary goals of the output team.
During the design of the output domain, we will use an unmodified version of CaM that will change conformation in the presence of calcium and the M13 peptide (as shown by Meister & Joshi, 2013). After optimizing our GLuc output domain, we will fuse it to the CaM input domain that the input team evolved to bind to our target peptide, ∂-toxin. This fused protein then should respond with luminescence to the presence of ∂-toxin rather than M13. We predict that even though these two domains were developed and optimized independently, they will work together in concert and in doing so, demonstrate the feasibility of moduler allosteric protein switches.
Project Goals:
- Create a GlucCaM that outputs G.Luc. chemiluminescence upon conformational change of CaM
- Optimize GlucCaM's dynamic range and gain
- Engineer GlucCaM to be reversible
