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| == Goals ==
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| # Find more than one pair of RNAIII/RNAII
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| # Asses:
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| ## Prove that these are orthogonal.
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| ## Improve the attenuation.
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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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| ** I asked Wes about this - basically it is statistically hard to have more than 1 plasmid per cell.
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| ==== Comments ====
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| * Anderson - 'If you think you can find what you want within a library of 10^5 clones, go with a lacZ /RFP/or GFP screen on plates...much easier to work out. If you need more diversity, your choices are an antibiotic selection or FACS. Both will be challenging to setup, but again the main hurdle in doing FACS is finding an available instrument, which might not even be an option.'
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| ==== 2-step selection method====
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| *I read through Chin's paper, and summarize the things need to do with this method.
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| ** [[User:Julius B. Lucks|Julius B. Lucks]] 00:37, 1 February 2008 (CST): I noticed in the acknowledgements that Chris Anderson provided them with the GH371 cells. We should ask Chris if this is a good cell line to do library selection in, and whether or not these people would be willing to give us a plasmid with their fusion pos-neg gene on it so we can pop it in our system.
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| *No matter screen or selection, we need 2 steps, one step is not enough to search for a switch.
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| [[Image:Constructs_StanleyQi.jpg]]
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| #Decide on best positive selection and negative selection methods. Assume their genes are ''pos'' and ''neg'' respectively. Obtain the plasmids containing each two genes.
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| #Choose certain E. coli strain which has least background expression of these two genes. Examine positive and negative selections separately.
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| #Construct fusion gene. Digest the two plasmids above and ligate them to modified pT181 or pIP501 plasmids (who no long express antisense RNAs any more) to give new plasmids: pT181f or pIP501f. Delete the leader mRNA region to generate a constitutive promoter pT181f+P (pIP501f+P). Mutate promoter region ?) to give a promoterless plasmid pT181f-P (pIP501f-P). Mutate the start codons to give a gene deletion plasmid pT181f+0 (pIP501f+0).
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| #* [[User:Julius B. Lucks|Julius B. Lucks]] 00:37, 1 February 2008 (CST): I think we could use Brantly and Wagner plasmids pGW501 and pGW181, which already have the promoter mutations and an RBS in there - we would just need to swap out the lacZ gene in there for the appropriate fusion pos-neg gene. We might consider bio-bricking some of the Brantl and Wagner plasmids so that we can swap reporter genes out easily. We would have to test if this changes the attenuation effeciency.
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| #Examine transformation of E. Coli under controls. Examine leaky transcription and background transcription.
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| #Enrichment studies. (?)
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| #Design target sites on antisense RNAs and leader mRNA sequences. Antisense RNAs are on another plasmid.
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| #Saturation mutagenesis (some PCR) to yield two libraries: antisense RNA library and leader mRNA library.
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| #Transform E. coli with leader mRNA lib, then apply positive selection.
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| #Transform surviving bacteria with antisense RNA lib, apply negative selection.
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| #Isolate plasmids from live bacteria colonies, sequence hits...
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