Biomod/2011/Slovenia/BioNanoWizards/methtimeline

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Timeline of our project


Before July

  • Team brainstorming sessions - lead theme: how can we make DNA origamis to work in applications? Position specific functionalization!
  • What tools and skills do we have: DNA origami (Vid), zinc fingers (Jernej), protein self assembly (Marko), AFM (Vid), recombinant DNA techniques (Marko, Jernej, Vid), recombinant protein production (Jernej, Marko, Vid), EMSA (Jernej), AlphaScreen (lab L12).
  • Cloning strategy selection & primer design for GST-ZFP, MBP-ZFP and RLuc-ZFP, YFP-ZFP ORFs (Jernej, Marko, Vid).
  • DNA origami design and verification of its assembly (Vid).

July


  • Cloning of ZFP chimeras into pET41a(+) vector (PCRs, agarose gel electrophoresis, isolation of PCR fragments from the gel, ligation, transformation, plasmid isolation, control restrictions, sequencing). Marko prepared GST-AZPA4, GST-6F6, GST-2C7, GST-Zif268, GST-sZif268 and GST-PBSII constructs, Vid prepared MBP-2C7 and MBP-6F6, Jernej prepared fusion proteins with either Renilla luciferase or yellow fluorescent protein mCitrine, namely RLuc-2C7 and YFP-AZPA4 as well as RLuc and YFP combinations with other ZFPs.
  • Transformation of sequence-verified constructs into production strain E. coli BL21(De3)pLysS and test productions. Analysis of cell culture lysate supernatants and precipitates (inclusion bodies) with SDS-PAGE and Western blot.

August


  • Production and isolation of GST-ZFP or MBP-ZFP fusions on Ni2+-NTA resin under the native conditions (Marko, Vid). ZFP fusions are soluble and well-behaved!
  • During all steps of isolation procedure protein purity was followed by SDS-PAGE and Western Blot.
  • Optimization of protein production conditions for RLuc-2C7 and YFP-AZPA4 chimeras to increase their solubility (variations in temperature, production media composition and IPTG concentration) (Jernej).

September


  • Binding of MBP-ZFP and GST-ZFP fusion proteins to DNA was followed by EMSA and AlphaScreen (Marko) and AFM (Vid). AlphaScreen assay works! ZFP-fusion proteins bind selectively to their target DNA sequences!
  • Production and isolation of additional MBP-ZFP and GST-ZFP fusions (Marko).
  • Design of primers for DNA tethers as well as cloning strategy outline for protein tethers, both to test vertical stacking of rectangular DNA origamis (Vid, Jernej).
  • First attempt to prepare vertical DNA stacks was half-successfull - 6 tethers resulted in a deformed stack, back to the drawing board.
  • Cloning of protein tethers (twin ZFPs with MBP solubility tag in between): 2C7-MBP-6F6, AZPA4-MBP-6F6 and AZPA4-MBP-2C7 (Jernej).
  • Design of primers and subsequent 3-point ligation cloning to obtain soluble MBP-RLuc-2C7 and MBP-YFP-AZPA4 BRET chimeras (Jernej).
  • Primer design for heterodimeric parallel and antiparallel coiled-coil as well as SH3 domain-SH3 ligand protein tethering constructs (they are planned to be the subject of the ongoing research, but we were running out of time to test them during the project) (Jernej).

October


  • Further binding studies followed by EMSA, AlphaScreen (Marko) and AFM (Vid). Vid detected specific binding of ZFP-fusions to DNA origami with AFM!
  • Production and isolation of protein tethers on Ni2+-NTA resins, 2C7-MBP-6F6 and AZPA4-MBP-6F6 (Jernej).
  • Assembly and observation of DNA-based vertical stacks using 10 DNA tethers after the introduction of the modified procedure (Vid), yes!!!
  • Production and isolation of MBP-RLuc-2C7 and MBP-YFP-AZPA4 on Ni2+-NTA resins, SDS-PAGE & Western blotting (Jernej). Both RLuc and YFP are functional for the BRET assay!
  • The last week was dedicated to refining and finalizing the WIKI content, movie and preparation of the presentation.
 

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