Difference between revisions of "Team:Bulgaria/Improve"

 
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{{Bulgaria}}
 
 
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<h1>Improve</h1>
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  width: 100%;
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
 
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
 
  
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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</div>
 
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 +
<div class="quickfind">
 +
  <button class="dropbtn">LEARN MORE</button>
 +
  <div class="quickfind-content">
 +
    <a href="#WHOUSE">PROJECT USE</a>
 +
<a href="#SAFE">IS IT SAFE</a>
 +
    <a href="#USEDSTRAINS">USED ORGANISM STRAINS</a>
 +
    <a href="#RISKS">POTENTIAL RISKS</a>
 +
<a href="#WASTE">WASTE TREATMENT</a>
 +
<a href="#EMERGENCY">EMERGENCY REACTIONS</a>
 +
<a href="#EQUIPMENT">PROTECTIVE EQUIPMENT</a>
 +
<a href="#OTHERS">OTHER RULES</a>
 +
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<a id="pagetitle">IMPROVE</a>
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 +
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<div id="scroldown"></div>
 +
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<!-- FOR NEW ONE COPY FROM HERE - CUSTOM CONTENT HOLDER -->
 +
<div class="contentHolder" id="PROJECTIMPROVE">
 +
<p>
 +
We improved the gRNA expression vector pSB1C3-gRNA - part BBa_K2515002
 +
from the Registry. It was originally designed by the iGEM Bulgaria 2017 team for easy
 +
cloning and expression of gRNAs in E. coli. Nevertheless, it is a high copy number vector
 +
with no easy option for a plasmid curing. To overcome these limitations, we PCR amplified
 +
the pSC101-based thermosensitive origin of replication from the pE-FLP vector (Plasmid
 +
#45978). Next, we developed an aqua cloning-based approach to substitute the original
 +
pSB1C3 origin with this new part. The resulting vector was low copy number (that is
 +
sufficient for gRNA expression due to the high efficiencies of the CRISPR/Cas9 systems)
 +
and can be eliminated by cultivation at 42 o C. </p>
 +
<p>
 +
Link: <a id="downlaodlink" href="http://parts.igem.org/Part:BBa_K2515002">http://parts.igem.org/Part:BBa_K2515002</a>
 +
<p>
 +
 +
NB! The generated vector is a low copy number plasmid, thus, one needs to use
 +
reduced antibiotic concentrations and the colony growth requires more time. In addition,
 +
the pSC101 ori contains a SpeI restriction site and, therefore, is not BioBrick RCF10-
 +
compatible! If you need to use it – adopt the aqua cloning procedure from the part’s page in
 +
the Registry. </p>
 +
<p>
 +
We submitted a new part BBa_K2847002 – it contains the thermosensitive replication
 +
origin and can be used as a template in PCR amplification reactions. </p>
 +
<p>
 +
Link: <a id="downlaodlink" href="http://parts.igem.org/Part:BBa_K2847002">http://parts.igem.org/Part:BBa_K2847002</a>
 +
</p></div>
 +
<!-- CUSTOM CONTENT HOLDER -->
 +
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 +
 +
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 +
<!-- CUSTOM CONTENT HOLDER -->
 +
<div class="contentHolder" id="second">
 +
<div style="font-size: 15px; margin-left: 70px; margin-right: 70px;">
 +
<strong>Fig. 1 pSB1C3-pSC101* T-sensitive origin. </strong><br>An illustration of our concept for a part
 +
improvement. The normal origin of replication found in pSB1C3 was substituted by the
 +
pSC101* T-sensitive origin. The resulting vector was a low copy number plasmid and
 +
required decreased antibiotic levels for growth (left). If needed, it could be successfully
 +
cured by an overnight cultivation at 42°C (right).
 +
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 +
</div>
 +
 +
<!-- TO HERE - CUSTOM CONTENT HOLDER -->
 +
 +
 +
<!-- CUSTOM MENU -->
 +
<div class="navigation" id="nav">
 +
  <a href="https://2018.igem.org/Team:Bulgaria">HOME</a>
 +
  <div class="dropdown">
 +
    <button class="dropNAVbtn">TEAM
 +
      <i class="fa fa-caret-down"></i>
 +
    </button>
 +
    <div class="dropdown-content">
 +
      <a href="https://2018.igem.org/Team:Bulgaria/theteam">MEMBERS</a>
 +
      <a href="https://2018.igem.org/Team:Bulgaria/attrib">ATTRIBUTIONS</a>
 +
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      <a href="https://2018.igem.org/Team:Bulgaria/theproject" >DESCRIPTION</a>
 +
      <a href="https://2018.igem.org/Team:Bulgaria/DESIGN">DESIGN</a>
 +
  <a href="https://2018.igem.org/Team:Bulgaria/PROTOCOLS">PROTOCOLS</a>
 +
  <a href="https://2018.igem.org/Team:Bulgaria/LABBOOK">LAB BOOK</a>
 +
  <a href="https://2018.igem.org/Team:Bulgaria/RESULTS">RESULTS</a>
 +
  <a href="https://2018.igem.org/Team:Bulgaria/CONTRIBUTION">CONTRIBUTION</a>
 +
  <a href="https://2018.igem.org/Team:Bulgaria/IMPROVE" id="activeMenu">IMPROVE</a>
 +
</div>
 +
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Latest revision as of 01:14, 18 October 2018

PROJECT USE IS IT SAFE USED ORGANISM STRAINS POTENTIAL RISKS WASTE TREATMENT EMERGENCY REACTIONS PROTECTIVE EQUIPMENT OTHER RULES

We improved the gRNA expression vector pSB1C3-gRNA - part BBa_K2515002 from the Registry. It was originally designed by the iGEM Bulgaria 2017 team for easy cloning and expression of gRNAs in E. coli. Nevertheless, it is a high copy number vector with no easy option for a plasmid curing. To overcome these limitations, we PCR amplified the pSC101-based thermosensitive origin of replication from the pE-FLP vector (Plasmid #45978). Next, we developed an aqua cloning-based approach to substitute the original pSB1C3 origin with this new part. The resulting vector was low copy number (that is sufficient for gRNA expression due to the high efficiencies of the CRISPR/Cas9 systems) and can be eliminated by cultivation at 42 o C.

Link: http://parts.igem.org/Part:BBa_K2515002

NB! The generated vector is a low copy number plasmid, thus, one needs to use reduced antibiotic concentrations and the colony growth requires more time. In addition, the pSC101 ori contains a SpeI restriction site and, therefore, is not BioBrick RCF10- compatible! If you need to use it – adopt the aqua cloning procedure from the part’s page in the Registry.

We submitted a new part BBa_K2847002 – it contains the thermosensitive replication origin and can be used as a template in PCR amplification reactions.

Link: http://parts.igem.org/Part:BBa_K2847002

Fig. 1 pSB1C3-pSC101* T-sensitive origin.
An illustration of our concept for a part improvement. The normal origin of replication found in pSB1C3 was substituted by the pSC101* T-sensitive origin. The resulting vector was a low copy number plasmid and required decreased antibiotic levels for growth (left). If needed, it could be successfully cured by an overnight cultivation at 42°C (right).