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Research Abstracts - 2007
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Engineering Synthetic Splicing Ribozyme Systems

Austin Che & Tom Knight

Synthetic Biology

The emerging field of synthetic biology aims to design and construct novel biological systems. The Knight lab is working on making it easier to engineer synthetic biological systems, drawing upon ideas from other engineering disciplines such as electrical engineering and computer science. Whereas electrical engineers have oscilloscopes and computer programmers have print statements, biologists have a more limited toolkit for debugging systems that they build. We are working to add programmable ribozymes that can modify existing RNA to the biologist's toolkit. Not only can these ribozymes be useful for biological debugging, the ribozymes can be used in building synthetic systems that would be difficult to build otherwise.


cis-splicing ribozymes
We chose the well studied Tetrahymena group I intron as the ribozyme core. The ribozyme naturally splices out of RNA and can be easily re-engineered to splice out of desired RNA.


trans-splicing ribozymes
By using relatively simple design rules and RNA base pairing, this natural self-splicing ribozyme can also be re-engineered for trans-splicing. In trans-splicing, the ribozyme targets an RNA of one's choice, replacing one part with another arbitrary RNA sequence.


This capability for introducing self-modifying code has the potential to be tremendously useful for the synthetic biologist. Splicing ribozymes enable the design of synthetic biological circuits by providing real-time control over what is expressed using post-transcription and pre-translation logic. The ability to splice into an existing biological system provides a minimally invasive hook into a running system for measurement and debugging purposes. In addition, splicing can be used to "patch" or modify the operation of an existing system. For building novel synthetic systems, splicing ribozymes can be used as a macro expansion library, expanding short tags to longer sequences. Another application is the implementation of logic using a modular, reusable, and scalable family of splicing ribozyme logic gates. For example, n-input AND gates could be built using only trans-splicing ribozymes without any translation. The primary practical concern in using these ribozymes is their splicing efficiency, but we are optimizing the activity of the ribozyme in the hope that it will become a basic tool for engineering biology.


[1] Brian G. Ayre and Uwe Köhler and Robert Turgeon and Jim Haseloff. Optimization of trans-splicing ribozyme efficiency and specificity by in vivo genetic selection. In Nucleic Acids Research, p. e141, 30(24):200.

[2] Jonghoe Byun and Ning Lan and Meredith Long and Bruce A. Sullenger. Efficient and specific repair of sickle β-globin RNA by trans-splicing ribozymes. In RNA, pp. 1254--1263, 9:2003.

[3] Uwe Köhler and Brian G. Ayre and Howard M. Goodman and Jim Haseloff. Trans-splicing ribozymes for targeted gene delivery. In Journal of Molecular Biology, pp. 1935--1950, 285(5):1999.

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