Difference between revisions of "Team:Vilnius-Lithuania/Protocols"

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<h1 class="text-wall-heading">Protocols</h1>
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<h1 class="text-wall-heading">Lab Book</h1>
 
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<div class="text-wall-area-box">
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     <h2 class="text-wall-area-box-heading">How to Repeat?</h2>
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     <h2 class="text-wall-area-box-heading">Writing a Book</h2>
 
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     <div class="scroll-area">
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         <p class="text-content">To achieve our goal, we used different methods which are described in respective protocols. We provide a detailed picture of experiment workflow that experiments could be accurately repeated. Protocols include the aim of each experiment, list of used reagents and materials, description, protocol and illustrative schemes of different devices and processes.</p>
+
         <p class="text-content">Our team intensively and precisely worked in the lab for 15 weeks from the beginning of summer till the middle of autumn to develop a SynDrop idea. Here is a detailed documentation of our Wet Lab work, including tasks, reagents used and conditions needed during the experiments.</p>
  
 
         </p>
 
         </p>
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         <h1>Protocols</h1>
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         <h1>Lab Book</h1>
 
         <p></p>
 
         <p></p>
 
         <p></p>
 
         <p></p>
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         <h3>Chip preparation</h3>
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         <h2></h2>
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            <object data="
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<object data="https://static.igem.org/mediawiki/2018/3/33/T--Vilnius-Lithuania--a._b._Microfluidics.pdf" type="application/pdf" width="100%" height="700px" internalinstanceid="5" title="">
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            <object data="https://static.igem.org/mediawiki/2018/8/81/T--Vilnius-Lithuania--Week_1_Labook.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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  <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/3/33/T--Vilnius-Lithuania--a._b._Microfluidics.pdf">click here to
+
              <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/8/81/T--Vilnius-Lithuania--Week_1_Labook.pdf">click here to
−
      download the PDF file.</a>
+
                  download the PDF file.</a>
 +
              </p>
 +
            </object>
 +
            <p></p>
 +
                <h3>
 +
                      Beta barrels assembly validation
 +
                </h3>" width="100%" height="700px" internalinstanceid="5" title="">
 +
              <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/e/ea/T--Vilnius-Lithuania--m._PROTOCOLS_competent_cells.pdf">click here to
 +
                  download the PDF file.</a>
 +
              </p>
 +
            </object>
 +
            <p></p>
 +
                <h3>
 +
                      Beta barrels assembly validation
 +
                </h3>
 +
        <p>The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:</p>
 +
        <ol>
 +
            <li>Selecting the number of plasmid groups</li>
 +
            <li>Choosing the copy number of each group</li>
 +
            <li>Picking the type of copy number control (specific to one group or regulating all of them at once).</li>
  
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  </p>
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        </ol>
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</object>
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        </p>
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<p> </p>
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<h3>Coating</h3>
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<object data="https://static.igem.org/mediawiki/2018/5/5b/T--Vilnius-Lithuania--c._PROTOCOLS_Coating.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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        <p></p>
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  <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/5/5b/T--Vilnius-Lithuania--c._PROTOCOLS_Coating.pdf">click here to
+
        <p>The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics
−
      download the PDF file.</a>
+
            (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low
−
  </p>
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            copy number plasmid groups are not lost during the division.
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</object>
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        </p>
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<p></p>
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        <p></p>
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    <h3 id='liposomes_preparation'>
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        <div class="img-cont">
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             Liposomes preparation
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            <img src="https://static.igem.org/mediawiki/parts/8/84/Collect.png" alt="img">
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    </h3>
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            <div class="img-label">
 +
            </div>
 +
        </div>
 +
        <h2>Applications</h2>
 +
        <p>
 +
            <h5>Everyday lab work</h5>
 +
            <p>
 +
                A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your
 +
                research to a particular plasmid copy number just because there are not enough right replicons to
 +
                choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that
 +
                suits your needs.</li>
 +
            </p>
 +
            <h5>Biological computing</h5>
 +
            <p>
 +
                The ability to choose a wide range of copy number options and their control types will make the
 +
                synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number
 +
                regulation is equivalent to adding a global parameter to a computer system. It enables the coordination
 +
                of multiple gene group expression.
 +
            </p>
 +
            <h5>Smart assembly of large protein complexes</h5>
 +
             <p>
 +
                The co-expression of multi-subunit complexes using different replicons brings incoherency to an already
 +
                chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses
 +
                the same type of control, and in addition can act in a group-specific manner.</p>
  
−
<object data="https://static.igem.org/mediawiki/2018/2/2b/T--Vilnius-Lithuania--d._PROTOCOLS_Liposomes_preparation.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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            <h5>Metabolic engineering</h5>
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    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/2/2b/T--Vilnius-Lithuania--d._PROTOCOLS_Liposomes_preparation.pdf        ">click here to
+
            <p>
−
      download the PDF file.</a>
+
                A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the
−
    </p>
+
                levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in
−
</object>
+
                transcriptional control and synthetic ribosome binding sites in translational control are already
−
<p></p>
+
                widely used to maintain expression levels. In addition to current approaches, our framework allows a
−
    <h3>
+
                simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the
−
          Ribosome modification
+
                search for perfect conditions a lot easier.
−
    </h3>
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−
+
−
<object data="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--e._PROTOCOLS_e.coli_genome_modif.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--e._PROTOCOLS_e.coli_genome_modif.pdf">click here to
+
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      download the PDF file.</a>
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    </p>
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</object>
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<p></p>
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    <h3>
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            Protein purification (BAM complex)
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    </h3>
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−
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−
<object data="https://static.igem.org/mediawiki/2018/1/1b/T--Vilnius-Lithuania--f._PROTOCOLS_Protein_purification.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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−
    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/1/1b/T--Vilnius-Lithuania--f._PROTOCOLS_Protein_purification.pdf">click here to
+
−
      download the PDF file.</a>
+
−
    </p>
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−
</object>
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−
<p></p>
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−
    <h3>
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            Thermoswitches characterization
+
−
    </h3>
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−
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−
<object data="https://static.igem.org/mediawiki/2018/e/ed/T--Vilnius-Lithuania--h._PROTOCOLS_Thermoswitches.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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−
    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/e/ed/T--Vilnius-Lithuania--h._PROTOCOLS_Thermoswitches.pdf">click here to
+
−
      download the PDF file.</a>
+
−
    </p>
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</object>
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<p></p>
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    <h3>
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            Cloning
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−
    </h3>
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−
+
−
<object data="https://static.igem.org/mediawiki/2018/e/ef/T--Vilnius-Lithuania--i._PROTOCOLS_Cloning.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/e/ef/T--Vilnius-Lithuania--i._PROTOCOLS_Cloning.pdf">click here to
+
−
      download the PDF file.</a>
+
−
    </p>
+
−
</object>
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−
<p></p>
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−
    <h3>
+
−
        Competent cells preparation
+
−
    </h3>
+
−
+
−
<object data="https://static.igem.org/mediawiki/2018/e/ea/T--Vilnius-Lithuania--m._PROTOCOLS_competent_cells.pdf" width="100%" height="700px" internalinstanceid="5" title="">
+
−
    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/e/ea/T--Vilnius-Lithuania--m._PROTOCOLS_competent_cells.pdf">click here to
+
−
      download the PDF file.</a>
+
−
    </p>
+
−
</object>
+
−
<p></p>
+
−
    <h3>
+
−
            Beta barrels assembly validation
+
−
    </h3>
+
−
+
−
<object data="https://static.igem.org/mediawiki/2018/8/84/T--Vilnius-Lithuania--k._PROTOCOLS_Folding_assay.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/8/84/T--Vilnius-Lithuania--k._PROTOCOLS_Folding_assay.pdf">click here to
+
−
      download the PDF file.</a>
+
−
    </p>
+
−
</object>
+
−
<p></p>
+
−
    <h3>
+
−
            Exposition evaluation in bacteria/liposomes
+
−
    </h3>
+
−
+
−
<object data="https://static.igem.org/mediawiki/2018/4/4c/T--Vilnius-Lithuania--l._PROTOCOLS_Expossition.pdf" width="100%" height="700px" internalinstanceid="5" title="">
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    <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2018/4/4c/T--Vilnius-Lithuania--l._PROTOCOLS_Expossition.pdf">click here to
+
−
      download the PDF file.</a>
+
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    </p>
+
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</object>
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−
+
  
 +
 +
 +
            </p>
 +
 +
 +
        </p>
 +
        <p>
 +
        </p>
 +
        <table style="width:100%">
 +
<thead>
 +
<td align='center'>Species sign in ODE system</td>
 +
<td align='center'>Species</td>
 +
<td align='center'>Initial concentration (M)</td>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td align='center'>A</td>
 +
<td align='center'>pDNA+RNA I+RNAII early</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>B</td>
 +
<td align='center'>pDNA+RNA II short</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>RNAI</td>
 +
<td align='center'>RNA I</td>
 +
<td align='center'>1E-6</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>D</td>
 +
<td align='center'>pDNA+RNA II long</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>E</td>
 +
<td align='center'>pDNA+RNAII primer</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>F</td>
 +
<td align='center'>RNA II long</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>G</td>
 +
<td align='center'>pDNA</td>
 +
<td align='center'>4E-8*</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>H</td>
 +
<td align='center'>pDNA+RNA II+RNA I late</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>RNA II</td>
 +
<td align='center'>RNA II</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
<tr>
 +
<td align='center'>J</td>
 +
<td align='center'>RNAI+RNAII</td>
 +
<td align='center'>0</td>
 +
</tr>
 +
</tbody>
 +
</table>
 
     </div>
 
     </div>
 
</div>
 
</div>

Revision as of 02:32, 18 October 2018

Lab Book

Writing a Book

Our team intensively and precisely worked in the lab for 15 weeks from the beginning of summer till the middle of autumn to develop a SynDrop idea. Here is a detailed documentation of our Wet Lab work, including tasks, reagents used and conditions needed during the experiments.

Lab Book

It appears you don't have a PDF plugin for this browser, you can click here to download the PDF file.

Beta barrels assembly validation

" width="100%" height="700px" internalinstanceid="5" title="">

It appears you don't have a PDF plugin for this browser, you can click here to download the PDF file.

Beta barrels assembly validation

The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:

  1. Selecting the number of plasmid groups
  2. Choosing the copy number of each group
  3. Picking the type of copy number control (specific to one group or regulating all of them at once).

The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low copy number plasmid groups are not lost during the division.

img

Applications

Everyday lab work

A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your research to a particular plasmid copy number just because there are not enough right replicons to choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that suits your needs.

Biological computing

The ability to choose a wide range of copy number options and their control types will make the synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number regulation is equivalent to adding a global parameter to a computer system. It enables the coordination of multiple gene group expression.

Smart assembly of large protein complexes

The co-expression of multi-subunit complexes using different replicons brings incoherency to an already chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses the same type of control, and in addition can act in a group-specific manner.

Metabolic engineering

A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in transcriptional control and synthetic ribosome binding sites in translational control are already widely used to maintain expression levels. In addition to current approaches, our framework allows a simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the search for perfect conditions a lot easier.

Species sign in ODE system Species Initial concentration (M)
A pDNA+RNA I+RNAII early 0
B pDNA+RNA II short 0
RNAI RNA I 1E-6
D pDNA+RNA II long 0
E pDNA+RNAII primer 0
F RNA II long 0
G pDNA 4E-8*
H pDNA+RNA II+RNA I late 0
RNA II RNA II 0
J RNAI+RNAII 0
invert