Difference between revisions of "IGEM:Caltech/2007/Project"

From OpenWetWare
Jump to navigationJump to search
(<center>Project Overview</center>)
(<center>Project Details</center>)
Line 36: Line 36:
* [[IGEM:Caltech/2007/Project/Recombineering|Recombineering]] the cro, N, and Q amber mutation genes into lambda zap
* [[IGEM:Caltech/2007/Project/Recombineering|Recombineering]] the cro, N, and Q amber mutation genes into lambda zap
*Design and testing of the [[IGEM:Caltech/2007/Project/Riboregulator|Riboregulator]]
*Design and testing of the [[IGEM:Caltech/2007/Project/Riboregulator|Riboregulator]]
*Titration curves for [[IGEM:Caltech/2007/Project/Cro|Cro]], [[IGEM:Caltech/2007/Project/N|N-antiterminator]], and [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]] to find out the percentage of bacterial lysis at different concentrations of aTc
*Titering [[IGEM:Caltech/2007/Project/Cro|Cro]], [[IGEM:Caltech/2007/Project/N|N-antiterminator]], and [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]] construct-containing bacterial to test construct-mediated rescue of lytic activity (in amber phage)
==<center>Current Status</center>==
==<center>Current Status</center>==

Revision as of 07:24, 26 October 2007

Caltech phage.jpg

iGEM 2007

Home        People        Project        Protocols        Notes        Changes       

Project Overview

The Caltech iGEM 2007 team is composed of four undergraduates from Caltech and one undergraduate from MIT. Team members are current juniors and seniors in biology, chemistry, chemical engineering, and biological engineering. The team was advised by three graduate students and three faculty mentors.

Our project tries to answer the following question: can viruses be engineered to selectively kill and/or integrate into specific subpopulations of target cells, based on their RNA or protein expression profiles? This addresses an important issue in gene therapy, where viruses engineered for fine target discrimination would selectively kill only those cells over or underexpressing certain disease or cancer associated genes. An even more ambitious goal would be to rewire target cells, by integrating a small gene cassette which would modify the target cell's expression profile, possibly fixing a disease state.

While this is clearly an ambitious goal, we managed to choose a simple model system for this problem suitable for undergraduates working over a summer. The bacteriophage λ is a classic, well studied virus capable of infecting E. coli, another classic model genetic sytem. We therefore seek to engineer a λ strain targeted to lyse specific subpopulations of E. coli based on their transcriptional profiles. Together, λ and E. coli provide a tractable genetic system for this larger problem, while hopefully providing lessons applicable to more ambitious, future projects.

Briefly, our project relies on controlling key viral developmental processes in a target-cell specific manner. In our design, the engineered viruses are capable of entering all cells. The viruses are engineered to lack the native copy of a key developmental gene, while containing a second, regulated, copy which is only expressed when the virus infects specific target cells. Thus, viruses infecting non-target cells stall early in their development and are quickly destroyed by the host. Viruses infecting target cells, however, manage to express these essential genes and successfully complete their infection cycle.

As an initial mechanism to target viruses to specific cell types, we will place the viral developmental genes under riboregulator control. Viral mRNAs for the regulated developmental genes will express with a stem loop sequestering ribosome binding sites, preventing translation. Specific mRNA in target E. coli will invade the stem loop, freeing the ribosome binding site and allowing proper translation. We believe this approach is more general than methods which might target specific cell-surface markers. Furthermore, if this method works, it would be possible in principle to extend viral mRNA regulation using aptamers capable of recognizing subtle signals such as post-translational modification.

We selected the viral developmental genes N, Q, and cro as promising targets for regulation. N and Q are antiterminators required for λ to transcribe its full set of genes. Viruses lacking these genes stall at extremely early developmental stages and are completely inviable, barely producing any viral mRNA. cro represents a potentially interesting means to bias whether the virus will lyse a target cell, or integrate into its DNA. This makes it an attractive candidate to investigate the rewiring applications mentioned above.

Choosing an appropriate λ strain poses a challenge. Existing strains with defective N, Q, and cro genes lack unique restriction sites to clone our constructs into. Therefore, we will utilize recombineering to introduce introduce these mutations into phages specifically designed to accept cloning inserts.

Working with lambda phage has been new and fun for all of us. We hope you take the time to browse our wiki and learn more about our work!

Caltech 2007 overview.gif

Introduction to Bacteriophage λ

Project Details

The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project:

Current Status

Currently, E. coli strains have been constructed that contain a low-copy plasmid construct where one of three key developmental viral genes - coding for the Cro, N, or Q proteins - is regulated by a tetracycline-dependent promoter. The addition of anhydrotetracycline (aTc) inactivates the tetracycline repressor and leads to the production of the respective viral protein in the E. coli cells. This allows us to control the concentration of viral protein produced in the cells by adding varying amounts of aTc to the bacterial growth media. Heterologous N and Q have been shown to complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q and prevented complementation. We were unable to express sufficient cro from a plasmid to rescue a cro mutant phage.

Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. Phages resulting from the recombineering process are also being screened for successful N and Q amber mutants.