Biomod/2013/Harvard/design

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==Input==
==Input==
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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. [[Image:Calmodulin2.jpg | right | 250 px | Calmodulin2‎]] 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.
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The goal of the input team is to improve the binding and switching activity of a β-lactamase-calmodulin, BlaCaM, fusion protein with respect to a previously non-functional analyte, <i>Staphylococcus aureus</i>δ-toxin. 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. [[Image:Calmodulin2.jpg | right | 250 px | Calmodulin2‎]] 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. [[Image:DirectedEvol.png | left | 300 px | Directed Evolution]] Directed evolution is a system of molecular engineering that makes use of natural selection to develop a molecular toward a specific activity. First, a library of mutant molecules is created from a molecule of interest. For a protein, library creation involves
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Directed evolution was performed by screening individual purified members of the library for activity toward a previously inactive peptide, <i>Staphylococcus aureus</i>δ-toxin. The Calmodulin portion of the BlaCaM fusion gene was mutagenized randomly using error-prone PCR 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 <i>in vitro</i>. 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. After as many rounds of diversification and screening as possible, the selected library members will exhibit binding to a previously unrecognized peptide.
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[[Image:DirectedEvol.png | left | 300 px | Directed Evolution]]
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Directed evolution was performed by screening individual purified members of libraries for activity toward a previously inactive peptide, <i>Staphylococcus aureus</i>δ-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 <i>in vitro</i>. 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. After as many rounds of diversification and screening as possible, the selected library members will exhibit binding to a previously unrecognized peptide.
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==Output==
==Output==

Revision as of 10:34, 11 October 2013

Design

Contents


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

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."

Modular Platform

Input

The goal of the input team is to improve the binding and switching activity of a β-lactamase-calmodulin, BlaCaM, fusion protein with respect to a previously non-functional analyte, Staphylococcus aureusδ-toxin. 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.
Calmodulin2‎
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.
Directed Evolution
Directed evolution is a system of molecular engineering that makes use of natural selection to develop a molecular toward a specific activity. First, a library of mutant molecules is created from a molecule of interest. For a protein, library creation involves

Directed evolution was performed by screening individual purified members of the library for activity toward a previously inactive peptide, Staphylococcus aureusδ-toxin. The Calmodulin portion of the BlaCaM fusion gene was mutagenized randomly using error-prone PCR 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. After as many rounds of diversification and screening as possible, the selected library members will exhibit binding to a previously unrecognized peptide.

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.

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.

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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