Biomod/2011/LMU/FolD'N'Assemble/Results: Difference between revisions
Timon Funck (talk | contribs) No edit summary |
Timon Funck (talk | contribs) |
||
(26 intermediate revisions by 2 users not shown) | |||
Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
<html> | |||
<style> | |||
#column-one {display:none; width:1000px;background-color: #0000ff;} | |||
#content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: auto;background-color: #ffffff; } | |||
</style> | |||
</html> | |||
<div style="text-align:center; float:left; margin-right:2em" > | <div style="text-align:center; float:left; margin-right:2em" > | ||
{| class="wikitable" cellpadding="15" | {| class="wikitable" cellpadding="15" | ||
Line 6: | Line 15: | ||
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Project|<span style="color:black;">'''THE PROJECT'''</span>]] | |style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Project|<span style="color:black;">'''THE PROJECT'''</span>]] | ||
|- | |- | ||
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/ | |style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Methods|<span style="color:black;">'''METHODS'''</span>]] | ||
|- | |- | ||
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Protocols|<span style="color:black;">'''PROTOCOLS'''</span>]] | |style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Protocols|<span style="color:black;">'''PROTOCOLS'''</span>]] | ||
|- | |- | ||
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/ | |style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Labbook|<span style="color:black;">'''LAB BOOK'''</span>]] | ||
|- | |||
|style="width:10em; border-top: 1pt black solid"| [[Biomod/2011/LMU/FolD%27N%27Assemble/Results|<span style="color:black;">'''RESULTS'''</span>]] | |||
|- | |- | ||
|style="width:10em; border-top: 1pt black solid"| '''TEAM''' | |style="width:10em; border-top: 1pt black solid"| '''TEAM''' | ||
Line 19: | Line 30: | ||
[[User:Ralf Weidner|<span style="color:black">Ralf Weidner</span>]] | [[User:Ralf Weidner|<span style="color:black">Ralf Weidner</span>]] | ||
[[User:Miranda Roßmann|<span style="color:black">Miranda Roßmann</span>]] | |||
[http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVschittler <span style="color:black">Verena Schüller</span>] | [http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVschittler <span style="color:black">Verena Schüller</span>] | ||
Line 24: | Line 37: | ||
[http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVliedl|<span style="color:black">Prof. Tim Liedl</span>] | [http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=CVliedl|<span style="color:black">Prof. Tim Liedl</span>] | ||
|- | |- | ||
|style="width:10em; height: | |style="width:10em; height:200em"| ''' ''' | ||
|} | |} | ||
</div> | </div> | ||
===Summary=== | ===Summary of achievements=== | ||
*The construct folded correctly. | *The construct folded correctly. | ||
*The cylinders dimerised correctly. The efficiency depends on the number of connections between the two cylinders | *The cylinders dimerised correctly. The efficiency depends on the number of connections between the two cylinders | ||
*The container openes at low pH depending on the salt concentration. | *The container openes at low pH depending on the salt concentration. | ||
*The opening also would work in human cells because it works in PBS buffer. | *The opening also would work in human cells because it works in PBS buffer. | ||
*The container can be loaded by adding payload to the annealing buffer. | |||
*Payload can be attached to the container by i-motif strands. | |||
===Folding=== | ===Folding=== | ||
As a first step, we designed the construct and looked for the best folding conditions. | As a first step, we designed the construct and looked for the best folding conditions. | ||
We chose a cylindrical shape with a closed and an open end. CaDNAno-file: | We chose a cylindrical shape with a closed and an open end. CaDNAno-file: http://openwetware.org/wiki/Image:Nanopill.json | ||
After some experiments, we decided to use a MgCl<sub>2</sub> concentration of 18 mM and an annealing time of 55 h. | After some experiments, we decided to use a MgCl<sub>2</sub> concentration of 18 mM and an annealing time of 55 h. | ||
The pictures of the TEM show that the cylinders have the right shape and size. | The pictures of the TEM show that the cylinders have the right shape and size (Fig. 1). | ||
[[Image:Tem_monomere.jpg]] | [[Image:Tem_monomere.jpg|thumb|center|500px|Fig.1: The monomers have the designed size and shape]] | ||
===Dimerisation=== | ===Dimerisation=== | ||
We designed four different constructs: with 2,3,4 and 5 pairs of complementary strands. [ | We designed four different constructs: with 2,3,4 and 5 pairs of complementary strands (Fig. 2-5). The red strands include the i-motif and the blue strands are the complementary strands. | ||
<center> | |||
{| | |||
||[[Image:Nanopill_querschnitt2.png|thumb|center|150px|4 connections]] | |||
||[[Image:Nanopill_querschnitt3.png|thumb|center|150px|6 connections]] | |||
||[[Image:Nanopill_querschnitt4.png|thumb|center|150px|8 connections]] | |||
||[[Image:Nanopill_querschnitt.png|thumb|center|150px|10 connections]] | |||
|} | |||
</center> | |||
More connection strands led to better dimerisation (Fig. 6). With 10 connections we almost got complete dimerisation. | |||
[[Image:Gel_dimerisation.jpg|thumb|center|500px|Fig. 6: 2% agarose gel. With more connection we get better dimerisation]] | |||
Pictures from the TEM show, that the dimerisation works correctly (Fig.7-8). | |||
[[Image:Tem dimersV.jpg|thumb|center|500px|Fig.7: Dimers with 10 connections]] | |||
[[Image:Tem dimereII.jpg|thumb|center|500px|Fig.8: Dimers with 4 connections]] | |||
===Opening=== | ===Opening=== | ||
To open the containers we did a buffer exchange. | To open the containers we did a buffer exchange. | ||
We exchanged the original buffer with another buffer with low pH. For good results we needed to decrease the salt concentration.[ | We exchanged the original buffer with another buffer with low pH. For good results we needed to decrease the salt concentration. With lower MgCl concentratione we got stronger monomer bands | ||
[[Image:Gel_saltproblem.jpg|thumb|center|400px|Fig.9: 2% agarode gel. The monomer bandes become stronger for lower salt concentrations ]] | |||
To show that the opening process would work in the human cell, we used PBS buffer (137 mM MgCl<sub>2</sub>, which is isotonic to the human body (Fig. 10). | |||
[[Image:Gel pbsopen.jpg|thumb|center|400px|Fig.10: 2% agarose gel. The samples with low pH have almost no dimers any more. The lanes with lower pH run slower because of the positive charge of the Hydrogen ions]] | |||
To check if the constructs survived the opening process we used the TEM again (Fig. 11). | |||
[[Image:Tem pbsopen.jpg|thumb|center|500px|Fig.11: Monomers after opening process]] | |||
===Loading by statistics=== | |||
After adding ssDNA+Cy5 to the annealing buffer we could show that the container is loaded (Fig. 12). | |||
[[Image:20110931.gif|thumb|center|500px|Fig. 12: Laser scanner (right) and florescence(left) image with Cy5 loaded dimers, 2% | |||
agarose gel, 3h running time]] | |||
===Loading by using i-motif strands=== | |||
To prove, that load could be attached by our i-motif sequence an a complementary sequence, we attached ssDNA with Cy5 to the conneection strands of our construct. In Fig. 13 we can see the release of the Cy5 at low pH. | |||
[[Image:20110924.gif|thumb|center|500px|Fig. 13: Laser scanner(left) and UV florescence(right) of Cy5 strands attached to the monomer, 2% agarose gel, 3h running time, left to right, monomers, monomers after buffer exchange pH 8, monomers after buffer exchange pH 4.5 ]] |
Latest revision as of 15:28, 2 November 2011
<html> <style>
- column-one {display:none; width:1000px;background-color: #0000ff;}
- content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: auto;background-color: #ffffff; }
</style> </html>
Summary of achievements
- The construct folded correctly.
- The cylinders dimerised correctly. The efficiency depends on the number of connections between the two cylinders
- The container openes at low pH depending on the salt concentration.
- The opening also would work in human cells because it works in PBS buffer.
- The container can be loaded by adding payload to the annealing buffer.
- Payload can be attached to the container by i-motif strands.
Folding
As a first step, we designed the construct and looked for the best folding conditions. We chose a cylindrical shape with a closed and an open end. CaDNAno-file: http://openwetware.org/wiki/Image:Nanopill.json
After some experiments, we decided to use a MgCl2 concentration of 18 mM and an annealing time of 55 h. The pictures of the TEM show that the cylinders have the right shape and size (Fig. 1).
Dimerisation
We designed four different constructs: with 2,3,4 and 5 pairs of complementary strands (Fig. 2-5). The red strands include the i-motif and the blue strands are the complementary strands.
More connection strands led to better dimerisation (Fig. 6). With 10 connections we almost got complete dimerisation.
Pictures from the TEM show, that the dimerisation works correctly (Fig.7-8).
Opening
To open the containers we did a buffer exchange.
We exchanged the original buffer with another buffer with low pH. For good results we needed to decrease the salt concentration. With lower MgCl concentratione we got stronger monomer bands
To show that the opening process would work in the human cell, we used PBS buffer (137 mM MgCl2, which is isotonic to the human body (Fig. 10).
To check if the constructs survived the opening process we used the TEM again (Fig. 11).
Loading by statistics
After adding ssDNA+Cy5 to the annealing buffer we could show that the container is loaded (Fig. 12).
Loading by using i-motif strands
To prove, that load could be attached by our i-motif sequence an a complementary sequence, we attached ssDNA with Cy5 to the conneection strands of our construct. In Fig. 13 we can see the release of the Cy5 at low pH.