IGEM:MIT/2009/OfficialWiki

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Welcome to the 2009 MIT iGEM wiki. This is our 6th year in competition since the inaugural iGEM competition in 2004. In the year of 2006, MIT's team won Best Systems and received 3rd place in the Presentations category. In 2008, MIT received a gold medal for their iGEM project. Hopefully, in 2009, MIT will become a finalist and win the iGEM competition with their exciting project.

Contents

Introduction

Biological switches are increasingly becoming a set of useful techniques. There are very few of these switches that are fast and reversible. Our project involves engineering baker's yeast, Saccharomyces cerevisiae, to be able to react to red and far-red light to be able to localize proteins to various points in the cell. This is done using the Phy-Pif system. Under exposure to red light, the proteins will begin to localize. Exposure to far red light will delocalize these proteins. This project has two components. One of these components involves metabolically engineering baker's yeast to be able to produce chromophores endogenously which are needed for this process to occur. The second part of this experiment is to localize portions of the Phy-Pif system to various points of the cell. Fluorescent proteins are used to observe the localization and delocalization of the proteins.

Project Overview

To maximize control over a biological system, it would beneficial to have quick, reversible control over each step in gene expression, from transcription to translation to post-translational processing. Much work has been done to create switchable promoters, toggled by pulses of light, to control rates of transcription for genes of interest. The MIT iGEM team aims to take this concept and apply it to post-translational control, more specifically protein targeting in yeast. Our goal is to make a system in which a pulse of light causes a protein of interest to localize to one part of the cell. When pulsed with another wavelength of light, the protein will diffuse. In this way, a user can easily control both localization and delocalization of a protein of interest.

Our system takes advantage of the machinery used by plants and algae to respond to changing light conditions. Small pigmented proteins called phytochromes allow plants and algae to sense the amount and quality of the light available to them and to adjust rates of transcription accordingly. They are composed of a small pigment called a chromophore covalently bonded to a polypeptide. PhyB, our phytochrome of interest, binds to a chromophore called phycocyanobilin, or PCB. In red light, phytochromes change conformation into it’s active form and can bind to a transcription factor called PIF3. A pulse of far-red light returns the phytochrome to its inactive state. This mechanism provides the foundation of a a fast, reversible switch.

We have two main goals for our project. Our first goal is to be able to engineer yeast to produce the chromophore PCB endogenously. Right now, PCB has to be extracted from plants or cyanobacteria or from strains of E. coli that have been designed to produce PCB. We would like the switch to be self-contained in the strain we engineer, so we would like to engineer a strain of yeast to produce PCB. Our second goal is to engineer a system that adopts the PhyB-PIF3 switch to control protein localization within the cell. We plan to have either PhyB or PIF3 constitutively anchored to the target desired (e.g. mitochondrial membrane, nuclear membrane, vacuole etc). The other will then be bound to our protein of interest and will diffuse within the cell. When pulsed with red light, the PIF3 and PhyB, causing the protein of interest to localize to the target.

Such a system would be useful in creating a reversible switch for synchronizing a culture of cells in one part of the cell cycle without having to deal with temperature sensitive mutants or adding chemicals externally to arrest the cell cycle. It would also be beneficial to study the kinetics of localization and delocalization, as well as provide an easy on-off switch for expression of essential genes. Light switchable transcriptional regulation has shown to be an effective way of increasing or decreasing gene expression quickly. By applying the concept of light switching to post-translational control as well, we aim to have greater control not only over gene expression, but also how the protein functions within the cell.

Members

Protocols

1. Extraction of PCB from Spirulina

2. Bacteria Transformation

3. Cloning Insert into Backbone

4. PCR Protocol

5. Yeast Transformation

6. Functional Assay for PCB

Progress

Link to Progress Calender

The PhyB-PIF3 System

Three main steps were taken to create a Reversible light switch in Yeast through the PhyB, PIF3 system.

1. Constructing the Plasmids

2. Testing Localization

3. Incorporating the PCB System

Metabolic Engineering of PCB Production In Yeast

1. Developing the Standard: PCB from Spirulina

2. The Second Leg in PCB Synthesis: HY2 and PcyA

3. The First Leg in PCB Synthesis: HMX1, HO1, HY1

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