Team:Queens Canada/ChimeriQ/Results

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As the goal of our project was to create chimeric flagella, the majority of our results were based around the creation of flagella with various inserts. In order to do so, we needed to make a mechanism by which we could easily create this chimeric primers. In doing so, we were able to create the part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781000"> K781000 </a>. 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. PCR overlap extension 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.   
As the goal of our project was to create chimeric flagella, the majority of our results were based around the creation of flagella with various inserts. In order to do so, we needed to make a mechanism by which we could easily create this chimeric primers. In doing so, we were able to create the part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781000"> K781000 </a>. 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. PCR overlap extension 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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<b>We are still in the process of obtaining results. What you see below is some initial testing of our parts. Sequencing data as well as additional motility assays and catalysis testing will soon follow.</b>
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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>
<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="thumb tleft"><div class="thumbinner" ><a href="/File:D3_RFP.JPG" class="image"><img alt="" src="/wiki/images/thumb/e/e7/D3_RFP.JPG/500px-D3_RFP.JPG" width="400" height="300" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:D3_RFP.JPG" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 4</b> The motility assay for the K781001 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct.</div></div></div>
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<div class="thumb tleft"><div class="thumbinner" style="width: 400px;" ><a href="/File:D3_RFP.JPG" class="image"><img alt="" src="/wiki/images/thumb/e/e7/D3_RFP.JPG/500px-D3_RFP.JPG" width="400" height="300" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:D3_RFP.JPG" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 1</b> The motility assay for the K781001 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct.</div></div></div>
<div class="thumb tright" width: 400px; float: right; margin-left: 0px; padding-right: 35px;><div class="thumbinner" style="width:422px;"><a href="/File:Rfp_flic_results_1.JPG" class="image"><img alt="" src="/wiki/images/thumb/9/9c/Rfp_flic_results_1.JPG/420px-Rfp_flic_results_1.JPG" width="420" height="272" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Rfp_flic_results_1.JPG" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 2:</b> TOP10 <i>E. coli</i> expressing J04450, the RFP insertion chimeric flagellin (K781001) and the RFP deletion chimeric flagellin (K781006).</div></div></div>
<div class="thumb tright" width: 400px; float: right; margin-left: 0px; padding-right: 35px;><div class="thumbinner" style="width:422px;"><a href="/File:Rfp_flic_results_1.JPG" class="image"><img alt="" src="/wiki/images/thumb/9/9c/Rfp_flic_results_1.JPG/420px-Rfp_flic_results_1.JPG" width="420" height="272" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Rfp_flic_results_1.JPG" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 2:</b> TOP10 <i>E. coli</i> expressing J04450, the RFP insertion chimeric flagellin (K781001) and the RFP deletion chimeric flagellin (K781006).</div></div></div>
<div class="thumb tleft"><div class="thumbinner" style="width:902px;"><a href="/File:Rfp_spectra.png" class="image"><img alt="" src="/wiki/images/thumb/b/b3/Rfp_spectra.png/900px-Rfp_spectra.png" width="900" height="283" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Rfp_spectra.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 3:</b> The fluorescence spectra of TOP10 <i>E. coli</i> cells expressing the RFP flagellin insertion <a href="http://partsregistry.org/Part:BBa_K781001" class="external text" rel="nofollow">K781001</a> and deletion <a href="http://partsregistry.org/Part:BBa_K781006" class="external text" rel="nofollow">K781006</a> constructs compared against <a href="http://partsregistry.org/Part:BBa_J04450" class="external text" rel="nofollow">J04450</a>, the corresponding RFP control.</div></div></div>
<div class="thumb tleft"><div class="thumbinner" style="width:902px;"><a href="/File:Rfp_spectra.png" class="image"><img alt="" src="/wiki/images/thumb/b/b3/Rfp_spectra.png/900px-Rfp_spectra.png" width="900" height="283" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Rfp_spectra.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 3:</b> The fluorescence spectra of TOP10 <i>E. coli</i> cells expressing the RFP flagellin insertion <a href="http://partsregistry.org/Part:BBa_K781001" class="external text" rel="nofollow">K781001</a> and deletion <a href="http://partsregistry.org/Part:BBa_K781006" class="external text" rel="nofollow">K781006</a> constructs compared against <a href="http://partsregistry.org/Part:BBa_J04450" class="external text" rel="nofollow">J04450</a>, the corresponding RFP control.</div></div></div>
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Our work with catalysis mainly involved working with three enzymes: LinB, Rv2579, and XylE. Using K781000, we were able to create three parts: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781005"> BBa_K781005 </a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781004"> BBa_K781004 </a>, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781007"> BBa_K781007 </a>. Based on the images below, it appears as if we were able to incorporate these enzymes into the bacteria, but more research much be done in order to characterize the enzyme activity. <br>
Our work with catalysis mainly involved working with three enzymes: LinB, Rv2579, and XylE. Using K781000, we were able to create three parts: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781005"> BBa_K781005 </a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781004"> BBa_K781004 </a>, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781007"> BBa_K781007 </a>. Based on the images below, it appears as if we were able to incorporate these enzymes into the bacteria, but more research much be done in order to characterize the enzyme activity. <br>
<div class="thumb tleft"><div class="thumbinner" style="width:402px;"><a href="/File:Rv2579_edit.png" class="image"><img alt="" src="/wiki/images/thumb/8/81/Rv2579_edit.png/500px-Rv2579_edit.png" width="400" height="300" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Rv2579_edit.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 4</b> The motility assay for the K781004 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.</div></div></div>
<div class="thumb tleft"><div class="thumbinner" style="width:402px;"><a href="/File:Rv2579_edit.png" class="image"><img alt="" src="/wiki/images/thumb/8/81/Rv2579_edit.png/500px-Rv2579_edit.png" width="400" height="300" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Rv2579_edit.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 4</b> The motility assay for the K781004 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.</div></div></div>
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<div class="thumb tleft"><div class="thumbinner" style="width:402px;"><a href="/File:Linb_mot.png" class="image"><img alt="" src="/wiki/images/thumb/f/fd/Linb_mot.png/500px-Linb_mot.png" width="400" height="300" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Linb_mot.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 4</b> The motility assay for the K781005 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.</div></div></div>
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<div class="thumb tleft"><div class="thumbinner" style="width:402px;"><a href="/File:Linb_mot.png" class="image"><img alt="" src="/wiki/images/thumb/f/fd/Linb_mot.png/500px-Linb_mot.png" width="400" height="300" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Linb_mot.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 5</b> The motility assay for the K781005 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.</div></div></div>
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Our work the Cohesin-Dockerin system had the ultimate goal of being able to create a chimeric flagella with a Dockerin insert, which would be able to bind with a specific Cohesin domain secreted by the bacteria. Unfortunately we were not able to reach this point, but were were able to create a part with  type II cohesin from Clostridium Thermocellum as a chimeric insertion into flagellin (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781003"> K781003 </a>).  
Our work the Cohesin-Dockerin system had the ultimate goal of being able to create a chimeric flagella with a Dockerin insert, which would be able to bind with a specific Cohesin domain secreted by the bacteria. Unfortunately we were not able to reach this point, but were were able to create a part with  type II cohesin from Clostridium Thermocellum as a chimeric insertion into flagellin (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K781003"> K781003 </a>).  
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<div class="thumb tleft"><div class="thumbinner" style="width:502px; style="width:400px; margin-left: 219px;"><a href="/Image:Cohesin_mot.png" class="image" title="Fig. 1 The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar."><img alt="Fig. 1 The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar." src="https://static.igem.org/mediawiki/2012/7/7a/Cohesin_mot.png" width="500" height="375" border="0" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/Image:Cohesin_mot.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 1</b> The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.</div></div></div>
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<div class="thumb tleft"><div class="thumbinner" style="width:502px; margin-left: 219px;"><a href="/Image:Cohesin_mot.png" class="image" title="Fig. 1 The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar."><img alt="Fig. 1 The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar." src="https://static.igem.org/mediawiki/2012/7/7a/Cohesin_mot.png" width="500" height="375" border="0" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/Image:Cohesin_mot.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div><b>Fig. 6</b> The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.</div></div></div>
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Latest revision as of 03:37, 27 October 2012
Control

ChimeriQ - Results

Overview
As the goal of our project was to create chimeric flagella, the majority of our results were based around the creation of flagella with various inserts. In order to do so, we needed to make a mechanism by which we could easily create this chimeric primers. In doing so, we were able to create the part K781000 . 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. PCR overlap extension 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.

We are still in the process of obtaining results. What you see below is some initial testing of our parts. Sequencing data as well as additional motility assays and catalysis testing will soon follow.
Fluorescence

The majority of our results fell under this section of our work as we were able to create both the RFP-FliC Insertion and deletion chimeras. These parts were 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.
    • Fig. 1 The motility assay for the K781001 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct.
    Fig. 2: TOP10 E. coli expressing J04450, the RFP insertion chimeric flagellin (K781001) and the RFP deletion chimeric flagellin (K781006).
    Fig. 3: The fluorescence spectra of TOP10 E. coli cells expressing the RFP flagellin insertion K781001 and deletion K781006 constructs compared against J04450, the corresponding RFP control.

    Catalysis
    Our work with catalysis mainly involved working with three enzymes: LinB, Rv2579, and XylE. Using K781000, we were able to create three parts: BBa_K781005 , BBa_K781004 , and BBa_K781007 . Based on the images below, it appears as if we were able to incorporate these enzymes into the bacteria, but more research much be done in order to characterize the enzyme activity.
    Fig. 4 The motility assay for the K781004 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.
    Fig. 5 The motility assay for the K781005 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.


    The Cohesin-Dockerin System
    Our work the Cohesin-Dockerin system had the ultimate goal of being able to create a chimeric flagella with a Dockerin insert, which would be able to bind with a specific Cohesin domain secreted by the bacteria. Unfortunately we were not able to reach this point, but were were able to create a part with type II cohesin from Clostridium Thermocellum as a chimeric insertion into flagellin ( K781003 ).
    Fig. 1 The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.
    Fig. 6 The motility assay for the K781003 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.
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