Biomod/2013/Harvard/design

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# Optimize GlucCaM's dynamic range and gain
# Optimize GlucCaM's dynamic range and gain
# Engineer GlucCaM to be reversible
# Engineer GlucCaM to be reversible
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[[Image:Gaussia Princeps.jpg|frame|none|The origin of GLuc - <em>Gaussia Princeps</em>]]
 
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Revision as of 14:17, 20 June 2013

Design

Contents

New Outputs‎


Calmodulin2‎

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.

Fig. 1: A Biosensor Enzyme (Adapted from   Mohanty et al, 2006)
Fig. 1: A Biosensor Enzyme (Adapted from Mohanty et al, 2006)

Modular Platform for Allosteric Switches

One type of biosensors is protein allosteric switches. Many proteins change conformation upon binding to a specific molecule, and these have been engineered so that conformational change due to binding activates

Allostry‎

"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."

Modular Platform

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. These conformational changes adjust the position of the two β-lactamase halves relative to each other, greatly affecting the activity of the enzyme. The ability to turn on or off the activity of the attached enzyme depending on the presence of an analyte gives the BlaCaM protein the ability to act as a sensor. By evolving BlaCaM to bind to different peptides or small molecules, the protein can be made into a sensor for a wide array of compounds. Adapting the BlaCaM switch is performed via directed evolution, where random mutations of the switch are screened and selected for increased effectiveness, and this process is iterated until a satisfactory new switch has been created.


Directed 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 GLucCam Protein in its Open State
The GLucCam Protein in its Open State

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.

Inorder 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.

The origin of GLuc - Gaussia Princeps
The origin of GLuc - Gaussia Princeps

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:

  1. Create a GlucCaM that outputs G.Luc. chemiluminescence upon conformational change of CaM
  2. Optimize GlucCaM's dynamic range and gain
  3. Engineer GlucCaM to be reversible
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