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
  • put 2 orthogonal ColE1s behind two genes - 2 diff inputs, make sure only one on for each input
• reconstruct collins switch
  • 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