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| | <div class="tabs-blue"> |
| | <ul> |
| | <li id="current">[[IGEM:Harvard/2006/DNA nanostructures|Project Overview]]</li> |
| | <li>[[IGEM:Harvard/2006/DNA_nanostructures/Designs|Designs]]</li> |
| | <li>[[IGEM:Harvard/2006/DNA_nanostructures/Notebook|Notebook]]</li> |
| | <li>[[IGEM:Harvard/2006/DNA_nanostructures/Protocols|Protocols]]</li> |
| | <li>[[IGEM:Harvard/2006/DNA_nanostructures/Presentations|Presentations]]</li> |
| | <li>[[IGEM:Harvard/2006/DNA_nanostructures/Literature|Literature]]</li> |
| | </ul> |
| | </div> |
| | <br style="clear:both"> |
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| ==Project Overview== | | ==Project Overview== |
| *Our goal is to to design and implement molecular containers, which can be dynamically opened and closed by an external stimulus. | | *Our goal is to design and implement molecular containers, which can be dynamically opened and closed by an external stimulus. |
| *The containers will be implemented as DNA nanostructures, which afford a significant degree of positional control and chemical versatility. | | *The containers will be implemented as DNA nanostructures, which afford a significant degree of positional control and chemical versatility. |
| *As an initial proof-of-concept, we plan to use our DNA containers to demonstrate controllable activation ("delivery") of anti-thrombin aptamers. | | *As an initial proof-of-concept, we plan to use our DNA containers to demonstrate controllable activation ("delivery") of anti-thrombin aptamers. |
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| **Cell sorting | | **Cell sorting |
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| ==Container Specs== | | ==Working Team Members== |
| [[Image:iGEM_harv06_mattspecs.gif]] | | *[[User:TChan|Tiffany Chan]] ([[User_talk:TChan|talk]], [[Special:Contributions/TChan|edits]]) |
| | *[[User:Kfifer|Katherine Fifer]] ([[User_talk:Kfifer|talk]], [[Special:Contributions/Kfifer|edits]]) |
| | *[[User:Vlau|Valerie Lau]] ([[User_talk:Vlau|talk]], [[Special:Contributions/Vlau|edits]]) |
| | *[[User:Matthewmeisel|Matthew Meisel]] ([[User_talk:Matthewmeisel|talk]], [[Special:Contributions/Matthewmeisel|edits]]) |
| | *[[User:Lhahn|Lewis Hahn]] ([[User_talk:Lhahn|talk]], [[Special:Contributions/Lhahn|edits]]) |
| | *TA: [[User:ShawnDouglas|Shawn Douglas]] ([[User_talk:ShawnDouglas|talk]], [[Special:Contributions/ShawnDouglas|edits]]) |
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| ==Container Designs== | | ==Recent Changes== |
| <gallery>
| | {{Special:Recentchanges/b=IGEM:Harvard/2006/DNA_nanostructures/&limit=20}} |
| Image:Igemharv06_Katie_Val_cylinderI.gif|[[IGEM:Harvard/2006/Container Design 1|Design 1]]<br>hexagonal core, separate 1-ply lids
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| Image:Smallcontainerdesign2.jpg|[[IGEM:Harvard/2006/Container Design 2|Design 2]]<br>hexagonal core, separate 2-ply lids
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| Image:Igemharv06_msmrect.png|[[IGEM:Harvard/2006/Container Design 3|Design 3]]<br>rectangular core, continuous 1-ply lids
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| Image:Websmallbarrsingleply.jpg|[[IGEM:Harvard/2006/Container Design 4|Design 4]]<br>hexagonal core, separate 1-ply lids
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| </gallery>
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| ==Latch Designs==
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| <gallery>
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| Image:iGEM_harv06_mattlatch1.jpg |latch1 <br>[[:Media:iGEM_harv06_mattlatch1.jpg|jpg]] | [[:Media:IGEM_harv06_mattlatch1.ai|ai]]
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| Image:iGEM_harv06_mattlatch2.jpg |latch2 <br>[[:Media:iGEM_harv06_mattlatch2.jpg|jpg]] | [[:Media:IGEM_harv06_mattlatch2.ai|ai]]
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| </gallery>
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| ==Coding==
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| ===Existing code===
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| *[[IGEM:Harvard/2006/DNA_nanostructures/Designing_DNA_nanostructures|William's code (Python)]]
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| ==Thrombin-aptamer experiments==
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| ====Questions / procedures====
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| * what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
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| * what incubation conditions?
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| * how much protein and DNA? protein at 1 {{um}}, DNA at 2 {{um}}
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| * Coomassie stain
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| | |
| ====Experiments====
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|number
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| | align="center" style="background:#f0f0f0;"|thrombin
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| | align="center" style="background:#f0f0f0;"|aptamer
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| | align="center" style="background:#f0f0f0;"|nanotube
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| | align="center" style="background:#f0f0f0;"|DNA-stained prediction
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| | align="center" style="background:#f0f0f0;"|protein-stained prediction
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| |-
| |
| |0||-||-||-||no bands||no bands
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| |-
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| |1||-||-||+||slow band (nanotube)||no bands
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| |-
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| |2||-||+||-||fast band (aptamer)||no bands
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| |-
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| |3||-||+||+||slow band (aptamer-nanotube), traces of fast band (aptamer)||no bands
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| |-
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| |4||+||-||-||no bands||fast band (thrombin)
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| |-
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| |5||+||-||+||slow band (nanotube)||fast band (thrombin)
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| |-
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| |6||+||+||-||medium band (aptamer-thrombin), fast band (aptamer)||medium band (aptamer-thrombin), traces of fast band (thrombin)
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| |-
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| |7||+||+||+||very slow band (thrombin-aptamer-nanotube), slow band (aptamer-nantotube), traces of fast band (aptamer)||very slow band (thrombin-aptamer-nanotube), medium band (aptamer-thrombin), traces of fast band (thrombin)
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| |-
| |
| |}
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| | |
| ====Buffers====
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| * Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub>
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| * Liu's incubation buffer: 40 mM Tris, 20 mM CH<sub>3</sub>COOH, 2mM EDTA, 12.5 mM (CH<sub>3</sub>COO)<sub>2</sub>Mg, pH 8.0
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| * Liu's PAGE buffer: 1x TAE/Mg<sup>2+</sup>
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| ====Protocols====
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| Potential protocol for a 2 {{ul}} incubation reaction (revised with Dr. Shih's suggestions)
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| * Reconsitute lyophilized [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/T6634 bovine thrombin]
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| ** biuret is 745 NIH units = 637 {{ug}} (1170 NIH units = 1 mg)
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| ** "A suggested concentration for preparation of a stock solution is 100 units/ml. The solution should contain approximately 0.1% BSA for stability and is stable for about one week at 0-5 °C. Since thrombin solutions adsorb to glass, it is recommended to aliquot the solution in plastic tubes and store at -20 °C or below." [http://www.sigmaaldrich.com/sigma-aldrich/product_information_sheet/t6634pis.pdf]
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| ** Reconstitute 745 NIH units in 0.49 mL 1% BSA and 4.41 mL water (4.90 mL total) to give a working stock of 152 units / mL = 130 {{ug}} / mL = 2 nmol / mL = 2 {{um}} (formula weight is approximately 65 kDa [http://www.sigmaaldrich.com/sigma-aldrich/product_information_sheet/t6634pis.pdf])
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| * In a 0.2 mL PCR tube, mix:
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| ** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
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| ** 1.0 {{ul}} of 2 {{um}} aptamers (final concentration: 1.0 {{um}} = 2 pmol)
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| ** 0.5 {{ul}} of 2 {{um}} thrombin (final concentration: 0.5 {{um}} = 1 pmol)
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| * OR in a 0.2 mL PCR tube, mix:
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| ** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
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| ** 0.5 {{ul}} of 2 {{um}} aptamers (final concentration: 1.0 {{um}} = 1 pmol)
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| ** 1.0 {{ul}} of 2 {{um}} thrombin (final concentration: 0.5 {{um}} = 2 pmol)
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| * Alternative mix: Liu uses 10 pmol of DNA (1 {{ul}} of 10 {{um}}) and varies thrombin amount from 2 pmol (1 {{ul}} of 0.2x thrombin working stock) to 100 pmol (1 {{ul}} of 10x thrombin working stock)
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| * Incubate at room temperature for 30 min.
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| * Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
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| ** Liu runs at 25 mA for 48 h.
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| [[User:Matthewmeisel|Matthewmeisel]] 11:11, 11 July 2006 (EDT)
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| | |
| ====Bibliography====
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| <biblio>
| |
| # tha1 pmid=8107090
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| # tha2 pmid=15945116
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| # tha3 pmid=8298130
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| # tha4 pmid=1741036
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| # tha5 pmid=8475124
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| </biblio>
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| ==Presentations==
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| ===Most recent (Week 3)===
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| * [[Media:IGEMHarv06 Week3 presentation VKTM2.ppt|Week 3 Presentation: Design Progress]]
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| ===Week 2: Original proposal===
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| * [[IGEM:Harvard/2006/DNA_nanostructures/Presentation_proposal|Presentation Proposal]]
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| ==Working Team Members==
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| *[[User:TChan|Tiffany Chan]] ([[User_talk:TChan|talk]])
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| *[[User:Kfifer|Katherine Fifer]] ([[User_talk:Kfifer|talk]])
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| *[[User:Vlau|Valerie Lau]] ([[User_talk:Vlau|talk]])
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| *[[User:Matthewmeisel|Matthew Meisel]] ([[User_talk:Matthewmeisel|talk]])
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| *...and others are welcome!
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