Difference between revisions of "Team:Madrid-OLM/AptDiscovery"
| Line 48: | Line 48: | ||
</section> | </section> | ||
| − | <section id=" | + | <section id="selex" class="text-center"> |
<div class="container"> | <div class="container"> | ||
<div class="row"> | <div class="row"> | ||
| Line 59: | Line 59: | ||
<p class="lead">With every round, more and more oligonucleotides with low binding-affinity for our protein of interest will be discarded leaving only strong binding aptamers at the end:</p> | <p class="lead">With every round, more and more oligonucleotides with low binding-affinity for our protein of interest will be discarded leaving only strong binding aptamers at the end:</p> | ||
<img alt="Image1" src="https://static.igem.org/mediawiki/2018/c/c3/T--Madrid-OLM--Aptamer--Discovery--Selexsquema.png" style="width:80%;"/> | <img alt="Image1" src="https://static.igem.org/mediawiki/2018/c/c3/T--Madrid-OLM--Aptamer--Discovery--Selexsquema.png" style="width:80%;"/> | ||
| + | |||
<h4>Our experiment:</h4> | <h4>Our experiment:</h4> | ||
<p class="lead">To separate the bound sequences from the unbound ones, we decided to use a nitrocellulose membrane.</p> | <p class="lead">To separate the bound sequences from the unbound ones, we decided to use a nitrocellulose membrane.</p> | ||
| Line 70: | Line 71: | ||
| − | + | <h4>BSA</h4> | |
| + | <p class="lead">We tested the detection limit of the ponceau dye to visualize the protein on the nitrocellulose membrane, which was 25 ug. This was decided to allow us to be able to detect, during the process, if the protein was being correctly binding with the membrane:</p> | ||
| + | <img alt="Image1" src="https://static.igem.org/mediawiki/2018/e/e7/T--Madrid-OLM--Aptamer--Discovery--BSA.png" style="width:80%;"/> | ||
| + | <p class="lead" style="margin-left:20%; margin-right:20%;">Table 1. It is shown the OD600 measurement by the first calibration experiment. The corrected Abs600 is made subtracting H2O measurements from LUDOX CL-X ones. The OD600/Abs600 result (3,652) is the multiplication factor, which you have to use after a cell density measurement with the plate reader to have a correct analysis.</p> | ||
| + | |||
</div> | </div> | ||
| Line 79: | Line 84: | ||
</section> | </section> | ||
| − | <section id=" | + | <section id="qPCR" class="text-center"> |
<div class="container"> | <div class="container"> | ||
<div class="row"> | <div class="row"> | ||
<div class="col-md-10 col-lg-8 boxed boxed--border bg--secondary boxed--lg box-shadow"> | <div class="col-md-10 col-lg-8 boxed boxed--border bg--secondary boxed--lg box-shadow"> | ||
| − | <h2> | + | <h2>qPCR</h2> |
<p class="lead"> | <p class="lead"> | ||
Cosas del apartado 1, se necesita meter mas divs seguramente | Cosas del apartado 1, se necesita meter mas divs seguramente | ||
Revision as of 22:04, 13 October 2018
Aptamer Discovery
Selex
SELEX: Systematic Evolution of Ligands via Exponential Selection is the process of identifying specific aptamers.
To select a binding aptamer, you don't look for epitopes. This only simplifies the process as you don't design a determined structure but reduced little by little an already binding population.
The SELEX screening process starts with a random library of nucleotide oligomers of a fixed size with know sequences in each end (for further amplification by PCR). Then the library is incubated with the target molecule. A number of this random sequences will bound to the target and the unbound sequences will be discharged.
The bound sequences are separated now from the target molecule (elution step) and amplified to create a new library.
With every round, more and more oligonucleotides with low binding-affinity for our protein of interest will be discarded leaving only strong binding aptamers at the end:
Our experiment:
To separate the bound sequences from the unbound ones, we decided to use a nitrocellulose membrane.
Nitrocellulose membranes can bind protein through hydrophobic interactions, so when applying the incubated library pool with the target protein to the membrane and forced the liquid to pass through it, the protein will stay within the membrane and as well the bound sequences.
The pore size was chosen after folding the aptamers and test both 0,22 um and 0.45 um size pore. After seeing the results we conclude that the 0.22 um pore was too small and the aptamers were being trapped because of their size and not by unspecifically interactions.
We tried in parallel two different proteins:
BSA
Ole-e1
BSA
We tested the detection limit of the ponceau dye to visualize the protein on the nitrocellulose membrane, which was 25 ug. This was decided to allow us to be able to detect, during the process, if the protein was being correctly binding with the membrane:
Table 1. It is shown the OD600 measurement by the first calibration experiment. The corrected Abs600 is made subtracting H2O measurements from LUDOX CL-X ones. The OD600/Abs600 result (3,652) is the multiplication factor, which you have to use after a cell density measurement with the plate reader to have a correct analysis.
qPCR
Cosas del apartado 1, se necesita meter mas divs seguramente