Team:TU-Delft/receptordesign

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<h2>Content</h2>  
<h2>Content</h2>  
<a href="#P1"> Chimeric receptor design: What, Why and How</a><br>
<a href="#P1"> Chimeric receptor design: What, Why and How</a><br>
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How: Align your final sequence to the original sequence of your receptor. Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br>
How: Align your final sequence to the original sequence of your receptor. Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br>
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br>
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br>
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<a name="P3"> <br><h2> Example</h2> </a><br>
<a name="P3"> <br><h2> Example</h2> </a><br>

Revision as of 10:07, 26 October 2012

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Receptor

Content

Chimeric receptor design: What, Why and How
In silico protocol
Example

Chimeric receptor design: What, Why and How

What?

Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7

Why?

One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].

How?

With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!


In silico protocol


  1. What: Get your receptor protein sequence code
    Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.
    How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file.
  2. What: look for the transmembrane regions
    Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.
    How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table. Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found ). When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes).
  3. What: Remove protein sequences that code for the N-terminal and C-terminal regions
    Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.
    How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right).
    Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.
  4. What: Check what you did so far
    Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions?
    How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.
  5. What: Go from protein sequence to DNA sequence
    Why: For further adaptations it is easier to work with the DNA sequence
    How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed.
  6. What: Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream
    Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.
    How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.
  7. What: Codon optimize the sequence for Saccharomyce cerevisiae
    Why: For better expression of the protein in yeast
    How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.
  8. What: add BamHI and NdeI restriction sites and other features
    Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.
    How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR.
    Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane.
  9. What: Final check
    Why: check, check, double check!
    How: Align your final sequence to the original sequence of your receptor. Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.
  10. Now you can send you sequence to a synthesizing company or work with isolated DNA.

Example