Written by: Julius Lucks.
Vipul Singhal, Kevin Spring, Shaima Al-Khabouri and Chris Fall – all students from the first CSHL SynBio Summer Course (2013) are authors on a recent paper on their work from the 2013 course.
We have been hypothesizing for a while that RNA-only circuits would be fast compared to protein circuits because RNAs degrade faster than proteins.
|To understand why the speed of genetic circuits is determined by the degradation rates of the signal carriers, see this excellent book by Uri Alon – “An Introduction to Systems Biology: Design Principles of Biological Circuits“.|
But up until now no one has been able to verify this. Led by Melissa Takahashi in the Lucks group, Vipul, Kevin, Shaima and Chris set out to lay the ground work for designing new experiments to do just this.
The goal of one of the CSHL SynBio modules in 2013 was to set up a system to use cell-free transcription-translation (TX-TL) reactions to rapidly characterize RNA-only genetic circuits. These circuits use special RNA mechanisms called transcription attenuators to regulate transcription. The trick is they do this based on the availability of another RNA molecule called an antisense RNA. Because they use an RNA input to control an RNA output, these mechanisms can be chained together to create ‘RNA-only’ genetic circuits. What we wanted to do is use the flexibility of TX-TL reactions to characterize the dynamics of RNA genetic circuitry to see how these systems really work.
TX-TL reactions contain all the molecular machinery necessary for the basic processes of gene expression, so they can express genetic circuits right from DNA templates. This makes them particularly fast for characterizing the properties of circuits – you can go from DNA to Data in a matter of a few hours, rather than a few days! In fact, Richard Murray (a visiting speaker at the Course in 2013 and 2014) and his group are creating a suite of experimental and computational tools so that Synthetic Biologists can use TX-TL as ‘Biomolecular Breadboard’ system for debugging genetic circuits.
The problem is that TX-TL systems were optimized for expressing proteins, so we didn’t know how they would work for RNAs. The research question set to Vipul, Kevin, Shaima and Chris in the course was: could TX-TL be used to characterize RNA-only genetic circuits? The answer they found was a resounding yes! Building off of their work in the course, we were able to design an experiment using the unique features of TX-TL to show that an RNA cascade propagates information at ~5 minutes per step, which is about the degradation half-life of one of our RNAs in this system. We also used some of the powerful tools of RNA engineering to show how you can tune this signal propagation time. This allowed us to make an RNA Single-Input Module, which is a network motif found in biology that is used to sequentially turn on one gene after another.
Along the way we learned a lot about using TX-TL to rapidly characterize the dynamics of RNA genetic circuitry, and we think that this system will be an important foundational tool for Synthetic Biology. There is still more work to be done to link circuit performance in TX-TL to circuit performance inside the cell, but we are already starting to make progress in that direction. One thing is for sure – something very exciting is bound to come out of the TX-TL RNA circuitry module at CSHL in 2014!
This work is also a testament to the vibrant spirit of the Cold Spring Harbor Synthetic Biology course, and a special thanks goes out to all of the instructors, speakers and students who made it happen!
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