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| = Stanley Li - Attenuation Project =
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| == Basic Idea ==
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| * want to make complicated logic gates in cells (for example)
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| * hard to do with proteins:
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| *# not enough orthogonal pairs
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| *# pairs that do exist have different kinetics (activities) and thermodynamics - makes it really hard to build complicated gates
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| == Questions ==
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| # Can we build a large orthogonal set of 'transcription factors'
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| # Can we have all of these be 'general' (same kinetics and thermodynaimcs)
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| == Antisense RNA system ==
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| * plasmid copy number control systems of pIP501 and pT181 (see Brantl and Wagner)
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| * looks like could be just antisense interaction
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| === Problems ===
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| * RNAs pretty big ('''how big?'''), so might be more than just secondary structure interaction
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| S:"50~300nt, diffusible, mostly untranslated, and highly structureed (one to four stem-loops))" (S. Brantl, 2007 Current opinion in
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| biology)
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| ** if a lot to do with tertiary structure, then might not be very designable
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| * attenuation low in E. coli (30%)
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| S:Why the attenuation is low in E. coli? Because of the nature of gram-negative bacteria? Is there any way to reduce the effect of
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| the host?
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| === Good Things ===
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| * all RNA, so hope of same thermodynamics and kinetics
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| * if only have to change small target sequence, could be designable
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| === Ideas about library construction ===
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| * 2 plasmids - one with RNAIII, one with RNAII
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| * do mutagenesis PCR
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| ** digest product and ligate into 2 diff backbones with 2 diff resistances
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| * mix together and transform mix
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| * select for both resistances
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| * screen
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| *# look for cells w/ no lacZ with RNAIII on
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| *# look for cells w/ lacZ with RNAIII off
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| * sequence hits
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| * using these, measure attenuation
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| ==== Problems/Questions ====
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| * need to estimate the size of the mutagensis 'space'
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| ** can only effectively screen a size of 10^4
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| * if make plasmids seperately, multiplying the number of possibilities - bad
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| * can we change the target region of RNAII without changing the embedded RNAIII and keep the same fold?
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| * How can we be sure to only put 1 plasmid of each type per cell?
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| == Goals ==
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| 0.) Repeat Brantl and Wagner experiments to verify that we can work with the system.
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| 1.) Find more than one pair of RNAIII/RNAII
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| 2.1) Prove that these are orthogonal.
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| 2.2) Improve the attenuation.
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| == TODOS ==
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| # Write protocols for step 1.
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| #* Which reporter (GFP, LacZ)?
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| #* RTPCR?
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| # Write down ideas for library construction and screening.
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| #* See paper on mRNA/RBS library design
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| # Test Kinefold (http://kinefold.curie.fr) to see if produces reliable results for our RNAs (also see Isambert, Siggia PNAS paper)
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| ## If this works, then try to do a computational study of the best target sites
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| == Other Questions ==
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| * Can RNAIII diffuse within the cell completely within its half life (<math>\tau</math>)?
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| ** Should be able to estimate this with <math>\sqrt{D\tau} \approx L \approx L_{cell} </math> - if this true, then we are fine
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