Julius B. Lucks/Meetings and Notes/01212008 Arkin

= ColE1 System - Engineerable transcriptional control =


 * idea - want to genericise transcriptional control
 * currently, theoretically, if want to build more complicated genetic networks, need more parts (promoters, terminators, etc.)
 * problems - each have their own kinetics
 * not necessarily orthogonal
 * to build something up, have to do massive characterization, then put together, then tweak to get to work together
 * parts proliferation problem
 * every gate in the system has to be made up of a different non-interacting promoter, etc.
 * still need to be tuned
 * one approach is the big FAB approach (Knight, Endy) -
 * build massive libraries of promoters and characterize them all in different cell contexts
 * then in multiple RBS contexts
 * then in multiple terminator contexts
 * might have enough characterization then to be able to build something up
 * HUGE cost, and not sure will work
 * probably something like will first do on 10 most popular things, then stop there
 * want to make the building of complicated system more amenable to predictable design
 * another approach via RNA engineering - translational control
 * Smolke - ribozymes - recognize metabolite - change conformation - allow translation
 * Benenson - RNAi governing transcript stability
 * these have been working in eukaryotes - mostly metazoans actually

ColE1 - RNA control of transcription via antisense interaction

 * what if we could use antisense interaction (like RNAi) to control transcritption
 * see image to understand the system (1)
 * ColE1 (72 bp region) - high copy number replication origin in E. coli (we actually use this in the lab)
 * when transcribed, forms some complicated secondary structure that allows polymerase to carry on, so ON by default
 * if certain antisense piece of RNA added, will bind to this, causing a DIFFERENT RNA secondary structure down the line, which does not allow polymerase to pass
 * natural NOT gate
 * true antisense mechanism - does not require other proteins to work
 * looks designable
 * could change the ColE1 sequence to recognize different key anti-sense sequences
 * hopefully be able to do this with simple anti-sense matching
 * hopefully be able to keep GC-content the same so that the thermodynamics would not change too much
 * want many different (so orthogonal) ColE1 regions so can put them together in different ways

Initial Targets

 * recreate original experiments
 * try to find a sequence with a stronger repression factor
 * prove orthogonal (2)
 * put 2 orthogonal ColE1s behind two genes - 2 diff inputs, make sure only one on for each input
 * reconstruct collins switch (3)
 * get one system to produce anti-sense of the other
 * to get this to work, would have to make sure the system is cooperative
 * would need to measure the induction curve - if sigmoidal, see what can do with it

Applications

 * since this is RNA mediated, doesn't matter where the RNA comes from
 * could come from cancer cells (which are known to over-express certain RNAs)
 * would have to design ColE1s to recognize these specific sequences
 * the best papers make a new type of part - more powerful - more computation power - some application for these cells
 * RNAi logic, Molecular turing machines - Kobi Benenson
 * lots of power
 * beyond Adelman
 * more towards what I want to do
 * figure out need computational power X to do thing Y - put in an application context

Problems

 * currently only about a 3-fold repression - not that much
 * may not be an anti-sense thing - could be something more complicated (Chris Anderson seemed to indicate)
 * could be that the dynamic range is poor
 * kinetics could be poor
 * could be good for a NOT gate, but might need more logic on the promoter (like promoters that have several inputs - do an integration right there)

TOREAD

 * talk to Anthony Carruthers (looking at changing mRNA degradation rates)
 * also Jonathan Golder
 * also David Tulga - works w/ Anderson - Xis and Int recombination systems
 * alos Chris Anderson about RNA genes
 * big ColE1 people in europe
 * Sabin Brandtl
 * Gerhard Wagner
 * which papers like and why - which ones are the most exciting

Random Notes

 * other systems kind of like this
 * SacT terminator of B. subtilis - sacT binds to hairpin and opens
 * anti-termination with no modifications of the polymerase
 * iGEM - FMN - translational control
 * were trying to avoid the protein part - just another step to worry abou engineering
 * Niles Pierce - multi-stranded RNA systems