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[[Image:NewOutput.png | 200 px | New Outputs‎]]




[[Image:Calmodulin2.jpg | 250 px | Calmodulin2‎]]
The modular approach naturally divides the project into two parts: the input domain and output domain.


==Biosensors==
==Input==


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.
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.  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 molecules is created from a molecule of interest. For a protein, library creation involves mutating the gene encoding the protein of interest and then expressing those mutant genes to create the library of proteins. This library is then screened for desired activity by hand or subjected to a selection that eliminates undesired mutations. [[Image:DirectedEvol.png | left | 300 px | Directed Evolution]]The proteins that survive the screen or selection can then have their encoding DNA sequenced to identify the beneficial mutations, or that DNA can be mutagenized into a new library, allowing a second generation of directed evolution to begin.  


[[Image:BiosensorEnzyme.png | frame | center | Fig. 1: A Biosensor Enzyme (Adapted from [[Biomod/2013/Harvard/References#General |  Mohanty et al, 2006]])]]
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.


==Modular Platform for Allosteric Switches==
==Output==


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


[[Image:Allostry.png | center | Allostry‎]]
===Overview===
The output team's goal is to alter the BlaCaM switch to produce a different output signal by replacing the transducer region of BlaCaM (split beta-lactamase) with another protein that will produce a different output signal in response to the conformation change of CaM. Among various forms of output signals--fluorescence, luminescence, electrical signal, enzymatic activity, etc.-- we chose luminescence for the relative ease in detection/visualization of luminescence, and furthermore, we chose to engineer ''Guassia princeps''-derived luciferase (GLuc) as the output domain protein to replace the beta-lactamase region of the existent BlaCaM. 


"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."
===GLuc and GLucCaM===
GLuc catalyzes the oxidation of the substrate coelenterazine to produce light of peak wavelength length around 480nm, as shown in the figure below:


[[Image:ModularPlatform.png | 650 px | center |Modular Platform]]
[[Image:GLucRxn.png|center|450px|alt = GLuc activity| Catalysis of luciferin coelentarazine]]


==Input==
Many features of GLuc distinguished GLuc as a highly attractive output domain for the project.  GLuc has a strong activity with its luminescence intensity upto 700-fold higher than that of Renilla or firefly-derived luciferases; it is highly stable, as it remains fully folded up to 40˚C and even retains 65% of its activity after 30 minutes of incubation at 95˚C; lastly, it is one of the smallest luciferase (20kD). 


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.
Moreover, GLuc is already gaining popularity as a reporter gene. For example, the following picture shows a in vivo imaging using GLuc as the reporter.


[[Image:GLucApp.png | 350px | thumb |center |An application of GLuc as a reporter (Adapted from [[Biomod/2013/Harvard/References#Output Domain |  Luker et al, 2012]])]]


[[Image:DirectedEvol.png | left | 300 px | 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.



[[Image:Gaussia Princeps.jpg|thumb|right|200px| alt = Gaussia Princeps| The origin of GLuc - <em>Gaussia Princeps</em>]]


Our goal was to replace the beta-lactamase output of BlaCaM with the GLuc output while keeping the CaM functionality intact.  To do so we replaced the beta-lactamase regions of BlaCaM with split GLuc to produce GLucCaM, a protein switch that activates GLuc activity in response to conformation change of CaM upon target binding.  Specifically, we sought to:


==Output==
# Create a GlucCaM that retains the chemiluminescence function of GLuc. 
# Explore different structure-activity relationships in GlucCaM
# Engineer GLucCaM to output chemiluminescence specifically in response to the target binding conformation changes of CaM.


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).


[[Image:GLucCaM Cartoon.png|thumb|left|250px|alt = GLucCam Protein| The GLucCam Protein in its Open State]] 
===Design of GLucCaM===


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 [[Biomod/2013/Harvard/References#Output_Domain | 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.  
GLuc will be split into two, yielding us the N-terminal and C-terminal halves of Gluc (N-GLuc and C-Gluc).  These two will replace the existing N-terminal and C-terminal halves of beta-lactamase in BLaCaM. We desire the GLucCaM switch to deactivate when CaM binds to Ca(2+) and assumes a rigid shape that separates the two halves of GLuc by distance, and then upon target binding, it should activate GLuc as CaM's conformation change brings the two halves into proximity.


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 first task is retaining the functionality of GLuc through the splitting.  The next task is to vary many parameters to test the function-structure relationship of the switch.  This step includes varying linker length, linker rigidity, mutations on Cysteine residues, expression conditions, etc. Some possible concrete challenges of this step are: 1) linkers may be too short or long, in which case the GLuc may never be active or constitutively active, 2) 10 disulfide bridges present in GLuc may prevent the two halves of GLuc from separating once they are brought to proximity, and 3) it has been noted that E.coli may not be the optimal expression medium for GLuc.


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 [[Biomod/2013/Harvard/References#General | 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.
The schematics of what we aim our final product to be be is as follows:


Project Goals:
[[Image:GLucCaMRxn.png|center|center|500px|alt = GLucCam Protein| The GLucCam Protein Schematics]]
# 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
[[Image:Gaussia Princeps.jpg|frame|none|The origin of GLuc - <em>Gaussia Princeps</em>]]
</div>

Latest revision as of 12:31, 26 October 2013

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Design


The modular approach naturally divides the project into two parts: the input domain and output domain.

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
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 is a system of molecular engineering that makes use of natural selection to develop a molecular toward a specific activity. First, a library of molecules is created from a molecule of interest. For a protein, library creation involves mutating the gene encoding the protein of interest and then expressing those mutant genes to create the library of proteins. This library is then screened for desired activity by hand or subjected to a selection that eliminates undesired mutations.
Directed Evolution
Directed Evolution
The proteins that survive the screen or selection can then have their encoding DNA sequenced to identify the beneficial mutations, or that DNA can be mutagenized into a new library, allowing a second generation of directed evolution to begin.

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

Overview

The output team's goal is to alter the BlaCaM switch to produce a different output signal by replacing the transducer region of BlaCaM (split beta-lactamase) with another protein that will produce a different output signal in response to the conformation change of CaM. Among various forms of output signals--fluorescence, luminescence, electrical signal, enzymatic activity, etc.-- we chose luminescence for the relative ease in detection/visualization of luminescence, and furthermore, we chose to engineer Guassia princeps-derived luciferase (GLuc) as the output domain protein to replace the beta-lactamase region of the existent BlaCaM.

GLuc and GLucCaM

GLuc catalyzes the oxidation of the substrate coelenterazine to produce light of peak wavelength length around 480nm, as shown in the figure below:

Catalysis of luciferin coelentarazine
Catalysis of luciferin coelentarazine

Many features of GLuc distinguished GLuc as a highly attractive output domain for the project. GLuc has a strong activity with its luminescence intensity upto 700-fold higher than that of Renilla or firefly-derived luciferases; it is highly stable, as it remains fully folded up to 40˚C and even retains 65% of its activity after 30 minutes of incubation at 95˚C; lastly, it is one of the smallest luciferase (20kD).

Moreover, GLuc is already gaining popularity as a reporter gene. For example, the following picture shows a in vivo imaging using GLuc as the reporter.

An application of GLuc as a reporter (Adapted from Luker et al, 2012)


The origin of GLuc - Gaussia Princeps

Our goal was to replace the beta-lactamase output of BlaCaM with the GLuc output while keeping the CaM functionality intact. To do so we replaced the beta-lactamase regions of BlaCaM with split GLuc to produce GLucCaM, a protein switch that activates GLuc activity in response to conformation change of CaM upon target binding. Specifically, we sought to:

  1. Create a GlucCaM that retains the chemiluminescence function of GLuc.
  2. Explore different structure-activity relationships in GlucCaM
  3. Engineer GLucCaM to output chemiluminescence specifically in response to the target binding conformation changes of CaM.


Design of GLucCaM

GLuc will be split into two, yielding us the N-terminal and C-terminal halves of Gluc (N-GLuc and C-Gluc). These two will replace the existing N-terminal and C-terminal halves of beta-lactamase in BLaCaM. We desire the GLucCaM switch to deactivate when CaM binds to Ca(2+) and assumes a rigid shape that separates the two halves of GLuc by distance, and then upon target binding, it should activate GLuc as CaM's conformation change brings the two halves into proximity.

The first task is retaining the functionality of GLuc through the splitting. The next task is to vary many parameters to test the function-structure relationship of the switch. This step includes varying linker length, linker rigidity, mutations on Cysteine residues, expression conditions, etc. Some possible concrete challenges of this step are: 1) linkers may be too short or long, in which case the GLuc may never be active or constitutively active, 2) 10 disulfide bridges present in GLuc may prevent the two halves of GLuc from separating once they are brought to proximity, and 3) it has been noted that E.coli may not be the optimal expression medium for GLuc.

The schematics of what we aim our final product to be be is as follows:

The GLucCam Protein Schematics
The GLucCam Protein Schematics
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