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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.  The first challenge will be to retain 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.  We desire the GLucCaM switch to deactivate when CaM binds to Calcium and assume a rigid shape that separates the two halves by distance, and upon target, it should activate GLuc as its conformation change brings the two halves together.  Some possible challenges 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, 3) it has been noted that E.coli may not be the optimal expression medium for GLuc.
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.  The first challenge will be to retain 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.  We desire the GLucCaM switch to deactivate when CaM binds to Calcium and assume a rigid shape that separates the two halves by distance, and upon target, it should activate GLuc as its conformation change brings the two halves together.  Some possible challenges 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, 3) it has been noted that E.coli may not be the optimal expression medium for GLuc.


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


[[Image:GLucCaMRxn.png|center|center|500px|alt = GLucCam Protein| The GLucCam Protein Schematics]]
[[Image:GLucCaMRxn.png|center|center|500px|alt = GLucCam Protein| The GLucCam Protein Schematics]]

Revision as of 16:51, 20 October 2013

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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.

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

Biosensors can be derived from many types of platforms. Perhaps the most well-known biosensor is the commonly used blood glucose monitor. This monitor measures glucose levels by detecting the product of glucose oxidase, hydrogen peroxide, with an electrode. This sensor relies on the natural enzyme glucose oxidase to convert glucose into products that can be easily quantified by electrodes. Such sensors rely on the enzymatic activity of proteins to amplify and convert a signal into forms that can be easily measured.

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
Modular Platform

Our starting point for this project is the BlaCaM protein, which is a β-lactamase-calmodulin fusion that exhibts the allosteric switching we hope to build upon. Upon binding to certain peptides, the central calmodulin domain of BlaCaM changes conformation and brings together two fragments of β-lactamase, activating the β-lactamase protein. When the peptide separates from the calmodulin section, β-lactamase splits apart again and activity disappears.

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).

The origin of GLuc - Gaussia Princeps

Our goal was to replace 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. The first challenge will be to retain 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. We desire the GLucCaM switch to deactivate when CaM binds to Calcium and assume a rigid shape that separates the two halves by distance, and upon target, it should activate GLuc as its conformation change brings the two halves together. Some possible challenges 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, 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

Project Goals

Input

  1. Develop a fast and efficient screening method for directed evolution of BlaCaM
  2. Engineer new peptide recognition into the BlaCaM protein switch via directed evolution
  3. Improve the specificity of this switch by also screening against activity to peptides with already known affinity

Output

  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.
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