Timeline of our project
- 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).
- 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.
- 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).
- 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).
- 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.