Team:Queens Canada/ChimeriQ/Parts

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<p>Guide to making Chimeric Proteins- Protein Expression</p>
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<p>ChimeriQ - Parts Page</p>
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<li> <a href="#Linkers"> Linkers </a> </li>
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<li> <a href="#Structures"> Structures on PDB</a> </li>
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<li> <a href="#Pymol">PYmol Building</a>  </li>
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<li> <a href="#Gromacs">GROMACS Simulations</a> </li>
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Parts Page
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Linkers
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This is the summary of some of our parts, these parts are a combination of our full flic constructs with different part insertions that have been tested. Our E.coli K12 FliC is our main part that consists of the full flic construct without the D3 domain variable. This part can be used to do part insertions within the sequence.
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<p>In our chimeras we have linker sequences on either side of the chimeric insert sequence. The linkers we chose to use were inert, and flexible linker sequences, with different sequences on either side so that overlap extension will be successful.
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Featured Parts
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<div class="contenttitle"> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781000 "> BBa_K781000 - [R0010][B0034] - E. coli K12 FliC
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<p>This is the complete flagellin coding sequence with biobrick standard cut sites removed, under the promoter R0010 and RBS B0034. This part can be used to over express the flagellin monomer.
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<h2>FliC -GGGGS- insert -GGSGG- FliC</h2>
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<p> It is important to also consider the flexible regions of the protein in which you are making your insertion. Flexible regions may be able to accommodate a protein insertion, and would not require very long linkers. However, longer linkers may be required to introduce enough flexibility for the inserted protein to fold properly.
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The main use of this part is for PCR overlap extension, which will make an insertion at the annotated site, given that the insert is in the appropriate format. The resulting chimera will express protein immediately after the PCR reaction is transformed.
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This part has been previously characterized as BBa_K777109. The part that we have submitted has some slight differences in the silent mutations made to remove Biobrick cut sites. This part also includes the promoter R0010 and the RBS B0034.
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<div class="contenttitle">  <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781001">BBa_K781001 - [R0010][B0034] - RFP-FliC Insertion Chimera
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Structures
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<p>The protein database is possibly the most important tool in chimeric protein design. Without having any previous knowledge of your structure, it is impossible to really say where or how you would design your chimeric protein.
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<p>This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion.
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In our case, the crystal structure for E. coli flagellin is not available on the protein database. However, the structure for the flagellin of S. typhymurium was available and is very similar to the structure of E. coli flagellin. The PDB ID is 1UCU.</p>
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<p>Additionally, some general assumptions can be made when searching for a good place to make an insertion. For example, the site that we chose to make our variable domain insertions is:</p>
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<p>This part was successfully characterized and determined to show expression of the red fluorescent protein from J04500. As can be seen below, there appears to be less detectable fluorescence in cell cultures expressing the different types of chimeras. This result can be expected for a number of possible reasons.</p>
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<h2>...AVTVANDGTVTMATG...</h2>
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<p>The insertion was made in between AVT and TVT, replacing the amino acid sequence VANDG. We needed to have some spacing in between the overlap regions for PCR overlap extension, which is why VANDG was replaced.</p>
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<li>   The chimeric protein may not be as stable as the free RFP.</li>
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<p>The presence of several threonine, alanine, valine and glycine residues is indicative of a loop region, which would make a good spot for an insertion. Additionally, when we used PCR to make the overlapping regions for the insertion, we amplified off of the already existing linker used for ovarlapping as a deletion. Hence our total linker was:
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<li>   The RFP may be polymerizing in flagella, resulting in a quenching effect as the fluorescent light may be absorbed by other RFP molecules arranged in close proximity.</li>
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<h2>...AVTTTGGGGS-insert-GGSGGTSTVT...</h2>
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<li>   The flagellin domains may hinder proper folding of the RFP. This may be why the more constrained deletion variant is showing less expression compared to the insertion variant, which has more flexibility. </li>
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<div class="contenttitle"> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781006">BBa_K781006 - [R0010][B0034] - RFP-FliC Deletion Chimera
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PYmol
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<p>First, you'll have to install it (don't worry, it's free): <a href="pymol.org">pymol.org</a></p>
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<p>Pymol is a really useful program for viewing and modifying protein structures. Here we'll quickly go step by step into specifically building proteins in PYmol and what you need to know</p>
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<li>Load your protein in PYmol: under 'plugins' choose 'PDB loader service' and enter the 4-digit PDB ID for your desired protein.
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<li>Right now you'll be in viewing mode, which lets you rotate and move the camera. To switch to "Editing mode" click on the box in the bottom right that reads "3-button viewing mode". This will switch you to editing mode.
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<li>Here you'll have all the options for actually moving your protein around and rotating it.
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<li>On the PYmol menu and script window, you can switch to "Builder". This will give you specific options for adding amino acids and other molecules to your structure.
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<li>To make an insertion, you'll have to delete a bond. This is one of the options in the Builder window. Or you can delete a residue or residues, by selecting a residue in viewer mode, right clicking and choosing remove.
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<li>Now you have free bonds where you can build residues. Simply click on the atom where you want to add a residue (usually a terminal N or C=O), then click on the residue you want to add in the Builder window. The default secondary structure is in a parallel beta sheet, which is essentially just a straight line.
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<li>Rotating bonds can be done by clicking on an atom and then double-click and hold the part that you want to rotate. Remember, don't rotate between N and C=O bonds!
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<li>The last and most important thing you'll need to know, which isn't obvious at first is how Pymol actually make bonds. It does it purely by proximity. So if you have atoms that are within 1.5 angstroms, Pymol will automatically make a bond between those two atoms after you save and reload the .pdb file.
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<li>The code "distance (pk1),(pk2)" will measure the distance between two selected atoms. You may also use the measurement wizard to find distances and well as phi and psi angles.
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<li>Move and twist your bonds and proteins to build your chimeric protein and then save them as one .pdb file!
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<p>This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as a deletion.
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<p>This part can be used as a template for making other PCR overlap insertions. After transforming the product, and digesting the template plasmid with Dpn1, any template that has not been successfully digested will form red colonies.</p>
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GROMACS
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<p>This part was successfully characterized and determined to show expression of the red fluorescent protein from J04500. As can be seen below, there appears to be less detectable fluorescence in cell cultures expressing the different types of chimeras. This result can be expected for a number of possible reasons.</p>
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<li>    The chimeric protein may not be as stable as the free RFP.</li>
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<li>    The RFP may be polymerizing in flagella, resulting in a quenching effect as the fluorescent light may be absorbed by other RFP molecules arranged in close proximity.</li>
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<li>    The flagellin domains may hinder proper folding of the RFP. This may be why the more constrained deletion variant is showing less expression compared to the insertion variant, which has more flexibility. </li>
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Submitted Parts
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<div class="contenttitle"> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781002">BBa_K781002 -  [R0010][B0034] - mgfp-5-FliC Insertion Chimera
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This is an mgfp-5 chimeric insertion flagella. mgfp-5 is a mussel foot binding protein.
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<div class="contenttitle">  <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781003">BBa_K781003 - [R0010][B0034] - Cohesin II-FliC Insertion Chimera
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This is type II cohesin from Clostridium Thermocellum as a chimeric insertion into flagellin.
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<div class="contenttitle">  <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781004">BBa_K781004 - [R0010][B0034] - Rv2579 Dehalogenase-FliC Insertion Chimera
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Rv2579 is a dehalogenase enzyme. This construct as incorporated that enzyme as an insertion into the variable domain.
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<div class="contenttitle">  <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781005">BBa_K781005 - [R0010][B0034] - LinB Dehalogenase-FliC Insertion Chimera
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LinB is a dehalogenase enzyme. It degrades haloalkanes which are toxic pollutants that accumulate in food chains. This part has the LinB incorporated at an insertion site.
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<div class="contenttitle">  <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781007">BBa_K781007 -  [R0010][B0034] - XylE-FliC Deletion Chimera
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This is an XylE deletion variant of the flagellin monomer. The enzyme XylE that degrades catechol was incorporated into the E. coli flagellin by replacing the variable domain.
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<div id='groupparts' style='min-height:100px;width:700px;  margin-left:auto; margin-right:auto;'><div style='width:300px;margin:2px;padding:20px;color:gray;border:1px solid gray'>Loading.....</div></div><script>$('#groupparts').load('/cgi/api/groupparts.cgi?t=iGEM012\&amp;g=Queens_Canada',function(){ $('#groupparts .tablesorter').tablesorter();} );</script>
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This is something that we are just getting into. Basically, what GROMACS does is simulates your protein structure or other molecule floating in a solvent (usually water). This can be really useful in estimating how your protein will actually behave with regards to its stability and flexibility. After running this you'll be able to see whether or not your enzyme's catalytic site might be facing the wrong way!
 
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Below is a link to a script, which contains a breakdown of each variable and how to actually do the run.
 
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<a href="https://static.igem.org/mediawiki/2012/e/e6/Run_md_gromos_cutoff.txt">Click here!</a> and then rename the script to a .sh instead of .txt.
 
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Latest revision as of 23:09, 26 October 2012

Control

ChimeriQ - Parts Page

Parts Page
This is the summary of some of our parts, these parts are a combination of our full flic constructs with different part insertions that have been tested. Our E.coli K12 FliC is our main part that consists of the full flic construct without the D3 domain variable. This part can be used to do part insertions within the sequence.

Featured Parts

This is the complete flagellin coding sequence with biobrick standard cut sites removed, under the promoter R0010 and RBS B0034. This part can be used to over express the flagellin monomer.

The main use of this part is for PCR overlap extension, which will make an insertion at the annotated site, given that the insert is in the appropriate format. The resulting chimera will express protein immediately after the PCR reaction is transformed.

This part has been previously characterized as BBa_K777109. The part that we have submitted has some slight differences in the silent mutations made to remove Biobrick cut sites. This part also includes the promoter R0010 and the RBS B0034.



This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion.

This part was successfully characterized and determined to show expression of the red fluorescent protein from J04500. As can be seen below, there appears to be less detectable fluorescence in cell cultures expressing the different types of chimeras. This result can be expected for a number of possible reasons.

  • The chimeric protein may not be as stable as the free RFP.
  • The RFP may be polymerizing in flagella, resulting in a quenching effect as the fluorescent light may be absorbed by other RFP molecules arranged in close proximity.
  • The flagellin domains may hinder proper folding of the RFP. This may be why the more constrained deletion variant is showing less expression compared to the insertion variant, which has more flexibility.


  • This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as a deletion.

    This part can be used as a template for making other PCR overlap insertions. After transforming the product, and digesting the template plasmid with Dpn1, any template that has not been successfully digested will form red colonies.

    This part was successfully characterized and determined to show expression of the red fluorescent protein from J04500. As can be seen below, there appears to be less detectable fluorescence in cell cultures expressing the different types of chimeras. This result can be expected for a number of possible reasons.

  • The chimeric protein may not be as stable as the free RFP.
  • The RFP may be polymerizing in flagella, resulting in a quenching effect as the fluorescent light may be absorbed by other RFP molecules arranged in close proximity.
  • The flagellin domains may hinder proper folding of the RFP. This may be why the more constrained deletion variant is showing less expression compared to the insertion variant, which has more flexibility.


  • Submitted Parts
    This is an mgfp-5 chimeric insertion flagella. mgfp-5 is a mussel foot binding protein.

    This is type II cohesin from Clostridium Thermocellum as a chimeric insertion into flagellin.

    Rv2579 is a dehalogenase enzyme. This construct as incorporated that enzyme as an insertion into the variable domain.

    LinB is a dehalogenase enzyme. It degrades haloalkanes which are toxic pollutants that accumulate in food chains. This part has the LinB incorporated at an insertion site.

    This is an XylE deletion variant of the flagellin monomer. The enzyme XylE that degrades catechol was incorporated into the E. coli flagellin by replacing the variable domain.



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