Biomod/2011/Slovenia/BioNanoWizards/methtimeline: Difference between revisions
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well as RLuc and YFP combinations with other ZFPs.</li> | well as RLuc and YFP combinations with other ZFPs.</li> | ||
<li>Transformation of sequence-verified constructs into | <li>Transformation of sequence-verified constructs into | ||
production strain E. coli BL21(De3)pLysS and test productions. Analysis | production strain <em>E. coli</em> BL21(De3)pLysS and test productions. Analysis | ||
of cell culture lysate supernatants and precipitates (inclusion bodies) | of cell culture lysate supernatants and precipitates (inclusion bodies) | ||
with SDS-PAGE and Western blot.</li> | with SDS-PAGE and Western blot.</li> | ||
| Line 529: | Line 529: | ||
<ul> | <ul> | ||
<li>Production and isolation of GST-ZFP or MBP-ZFP fusions on | <li>Production and isolation of GST-ZFP or MBP-ZFP fusions on | ||
Ni<sup>2+</sup>-NTA resin under the native conditions (Marko, Vid). ZFP fusions are | |||
soluble and well-behaved! | soluble and well-behaved! | ||
</li> | </li> | ||
| Line 578: | Line 578: | ||
Vid detected specific binding of ZFP-fusions to DNA origami with AFM! | Vid detected specific binding of ZFP-fusions to DNA origami with AFM! | ||
</li> | </li> | ||
<li>Production and isolation of protein tethers on | <li>Production and isolation of protein tethers on Ni<sup>2+</sup>-NTA | ||
resins, 2C7-MBP-6F6 and AZPA4-MBP-6F6 (Jernej).</li> | resins, 2C7-MBP-6F6 and AZPA4-MBP-6F6 (Jernej).</li> | ||
<li>Assembly and observation of DNA-based vertical stacks using | <li>Assembly and observation of DNA-based vertical stacks using | ||
10 DNA tethers after the introduction of the modified procedure (Vid), | 10 DNA tethers after the introduction of the modified procedure (Vid), | ||
yes!!!</li> | |||
<li>Production and isolation of MBP-RLuc-2C7 and MBP-YFP-AZPA4 | <li>Production and isolation of MBP-RLuc-2C7 and MBP-YFP-AZPA4 | ||
on | on Ni<sup>2+</sup>-NTA resins, SDS-PAGE & Western blotting (Jernej). | ||
Both RLuc and YFP are functional for the BRET assay! | Both RLuc and YFP are functional for the BRET assay! | ||
</li> | </li> | ||
Latest revision as of 15:36, 2 November 2011
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</ul> <!-- End PureCSSMenu.com MENU --> </div> </div> </div> <!-- end #header --> <div id="page" class="container"> <div id="content"> <div class="post"> <div class="entry"><big><big><big><big><span
style="color: black; font-weight: bold;">Timeline of our
project</span></big></big></big></big><br> <br> <span style="font-family: Arial;"><br> </span><big style="font-family: Arial;"><big><span
style="color: black; font-weight: bold;">Before July</span></big></big><br>
<span style="font-family: Arial;"><br> </span> <ul>
<li>Team brainstorming sessions - lead theme: how can we make
DNA origamis to work in applications? Position specific functionalization!</li>
<li>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). </li>
<li>Cloning strategy selection & primer design for
GST-ZFP, MBP-ZFP and RLuc-ZFP, YFP-ZFP ORFs (Jernej, Marko, Vid).</li>
<li>DNA origami design and verification of its assembly (Vid).</li>
</ul> <big style="font-family: Arial;"><big><span
style="color: black; font-weight: bold;"><br>
July</span></big></big><br> <span style="font-family: Arial;"><br> </span> <ul>
<li>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.</li>
<li>Transformation of sequence-verified constructs into
production strain <em>E. coli</em> BL21(De3)pLysS and test productions. Analysis of cell culture lysate supernatants and precipitates (inclusion bodies) with SDS-PAGE and Western blot.</li> </ul> <big style="font-family: Arial;"><big><span
style="color: black; font-weight: bold;"><br>
August</span></big></big><br> <span style="font-family: Arial;"><br> </span> <ul>
<li>Production and isolation of GST-ZFP or MBP-ZFP fusions on
Ni<sup>2+</sup>-NTA resin under the native conditions (Marko, Vid). ZFP fusions are soluble and well-behaved!
</li> <li>During all steps of isolation procedure protein purity was
followed by SDS-PAGE and Western Blot.</li>
<li>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).</li> </ul> <big style="font-family: Arial;"><big><span
style="color: black; font-weight: bold;"><br>
September</span></big></big><br> <span style="font-family: Arial;"><br> </span> <ul>
<li>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!
</li> <li>Production and isolation of additional MBP-ZFP and GST-ZFP
fusions (Marko).</li>
<li>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).</li>
<li>First attempt to prepare vertical DNA stacks was
half-successfull - 6 tethers resulted in a deformed stack, back to the drawing board.</li>
<li>Cloning of protein tethers (twin ZFPs with MBP solubility
tag in between): 2C7-MBP-6F6, AZPA4-MBP-6F6 and AZPA4-MBP-2C7 (Jernej).</li>
<li>Design of primers and subsequent 3-point ligation cloning
to obtain soluble MBP-RLuc-2C7 and MBP-YFP-AZPA4 BRET chimeras (Jernej).</li>
<li>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).</li> </ul> <big style="font-family: Arial;"><big><span
style="color: black; font-weight: bold;"><br>
October</span></big></big><br> <span style="font-family: Arial;"><br> </span> <ul>
<li>Further binding studies followed by EMSA, AlphaScreen
(Marko) and AFM (Vid). Vid detected specific binding of ZFP-fusions to DNA origami with AFM!
</li> <li>Production and isolation of protein tethers on Ni<sup>2+</sup>-NTA
resins, 2C7-MBP-6F6 and AZPA4-MBP-6F6 (Jernej).</li>
<li>Assembly and observation of DNA-based vertical stacks using
10 DNA tethers after the introduction of the modified procedure (Vid), yes!!!</li>
<li>Production and isolation of MBP-RLuc-2C7 and MBP-YFP-AZPA4
on Ni<sup>2+</sup>-NTA resins, SDS-PAGE & Western blotting (Jernej). Both RLuc and YFP are functional for the BRET assay!
</li> <li>The last week was dedicated to refining and finalizing the
WIKI content, movie and preparation of the presentation.</li> </ul> <span style="font-family: Arial;"><!-- TU SE KONEA GLAVNO BESEDILO NA STRANI --> </span></div> </div> </div> <!-- end #content --> <div style="clear: both;"> </div> </div> <!-- end #page --></div> <div id="footer-content" class="container"> <div id="footer-bg"> <table
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