Team:Peking R/Parts

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  <p class="notbookmaintitle" align=center>Softcoding of Genetic Program<a name="start" id="start"></a></p>
 
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    <p class="asdf">Parts of iGEM 2011 Peking_R</p>
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    <p class="aca">Favorite Parts</p>
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    <p><strong>1.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598000">BBa_K598000</a></strong></p>
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    <p><em>TPP  Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015</em></p>
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    <p> This part is TPP up-regulated hammerhead ribozyme, consisting of 131 nucleotides. </p>
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    <p>&nbsp;</p>
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     <p><strong>2.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598001">BBa_K598001</a></strong></p>
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     <p><em>Theophyline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator</em></p>
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    <p> This is a GFP generator regulated by theophylline responsive riboswitch. mRNA with a theophylline riboswitch in it responds to theophylline concerntration, producing different fluorescence strength of GFP. Regulated by different promoters, it would be used to demonstrate theophylline responsive curve, providing data for modeling. </p>
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    <p>&nbsp;</p>
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    <p><strong>3.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598002">BBa_K598002</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598002"></a></p>
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     <p><em>Bistable Switch Mutant 68</em></p>
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     <p> This part is one of the mutation libraries of bistable switch modifying the ribosome binding site (RBS) of <em>cI434</em> gene. </p>
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  <p class="mainbody"><strong>Background of softcoding</strong><br />
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     There are basically two design principles  in computer programming: hardcoding and softcoding. Hardcoding1  refers to the practice of embedding parameters and functions into the source  code of a program, whereas softcoding2 obtains values and functions  from external source. Hardcoding would be convenient when no dynamic parameters  are required in the program, but the source code should be rewritten anytime  the input data or functions change. On the contrary, softcoding enables users  to customize the software to their needs by altering external input, without  having to edit the program&rsquo;s source code time after time. </p>
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  <p class="mainbody">In the exciting field of synthetic biology,  various genetic programs have been developed to perform certain functions in  living organismswhich are similar to computer programs. For instance, a genetic  toggle switch <em>in vivo</em> was developed that could be switched between two states by chemical or thermal induction.Another  example is that an oscillatory network was constructed in which the synthesis  of green fluorescent protein was periodically induced.Yet  genetic programs need optimization to achieve ideal performance, especially  when several genetic modules are coupled. Similarly, optimization of metabolic  pathways is also a key issue in the field of metabolic engineering. </p>
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  <p>Traditional methods to optimize genetic  programs or metabolic pathways generally involve construction of libraries that  contain large amounts of mutants, and multi-round screening is usually  required. Apart from the obvious drawback that the constructing and screening  procedures are laborious and time-consuming, these methods could only generate  mutants with a fixed configuration, and to fine-tune their performance would  require another round of mutagenesis and selection, which resembles &ldquo;hardcoding&rdquo;.  Therefore, a platform for &ldquo;softcoding&rdquo; of genetic programs is urgently needed. </p>
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  <p>&nbsp;</p>
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  <p class="mainbody"><strong>Softcoding Approach</strong><br />
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  Based on the principle of softcoding, this  year our team established an extensible and versatile platform for softcoding  of genetic program, which is composed of anRNA toolkit and a methodology-- The RNA  toolkit consists of interoperable and truly modular ligand-responsive riboswitches/ribozymes, while the methodology is automated design of synthetic  ribosomal binding sites (RBS) with customized translation rate. When combining  them together, a quantitative correlation between the concentration of specific  ligand and synthetic RBS&rsquo; translation strength can be established. Therefore,  when tuning genetic program, customized RBS&rsquo; translation strength at multiple  sites can be high-throughputly achieved without having to conduct laborious  mutagenesis and characterization, followed by easily determining the  configuration of RBS(s)&rsquo;translation strength. Then RBS sequences that meet this  configuration will be automatically designed via computer  algorithms. </p>
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  <p class="mainbody">&nbsp;</p>
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    <em>RNA toolkit</em><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit">(Learn more...)</a><br />
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    Recently, RNA devices have emerged as powerful  tools to regulate gene expression <em>in vivo</em>,  and particularly, ligand-responsive riboswitches/ribozymes enable us to  manipulate translation strength of specific genes upon different concentrations  of ligands.<a href="#_edn3" name="_ednref3" title="" id="_ednref3"> </a>Ligand-responsive  riboswitches/ribozymes regulate the translation rate of downstream gene by changing  conformations, cleaving or splicing upon external addition of ligand. Compared  with transcriptional and post-translational regulation, riboswitches/ribozymes  function through allostery of RNA structure, which requires little or no  assistance from proteins, so the regulation mechanism is relatively simpler and  their functions are more decoupled from native biological activities.</p>
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<p class="mainbody">We characterized some existing  riboswitches/ribozymes, namely thiamine pyrophosphate (TPP)-responsive hammerhead  ribozymes and theophylline riboswitches. By altering the upstream promoter and  downstream coding sequence of the RNA controllers, we demonstrated that their  performance was independent from sequence context, which proved the modularity  of these RNA devices.</p>
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  <p class="mainbody">To further extend the range of application  of our RNA toolkit, we created a ribozyme that functions with a different mechanism, which has an extreme low level of backgrounds. We substituted the  aptamer domain of c-di-GMP group Intron to theophylline-responsive aptamer, thus  invented a group I intron that senses theophylline to perform splicing function.  Moreover, we introduced a general method to evolve hammerhead ribozyme that  senses a new ligand. By coupling an adenine aptamer with hammerhead ribozyme  and randomizing nucleotides in the linker domain, we evolved new hammerhead  ribozymes through dual selection, whose self-cleavage could be regulated by  adenine. Our project provided a new design principle for rational or  semi-rational design of riboswitches/ribozymes. </p>
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  <p class="mainbody"><em>RBS automated design</em><a href="https://2011.igem.org/Team:Peking_R/Project/RBSAutomatedDesign">(Learn more...)</a><br />
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  In bacteria, ribosomal binding site (RBS) sequence  is one of the most important determinants of translational initiation/translation  strength. Therefore manipulating RBS sequence would significantly affect the  translation strength of downstream gene. Salis <em>et.al</em>. used Gibbs energy (∆G) of the &ldquo;docked&rdquo; state of the mRNA-30S ribosomal subunit complex to  predict the translation strength of RBS sequence. Based on their pioneering  work, we developed a methodology that correlated the performance of the RNA  controllers under certain concentration of ligand to translation strength met  by corresponding RBS sequence. Combining this methodology with our RNA toolkit,  we can generate an RBS sequence through automated design once we achieved an  ideal configuration of genetic programs through RNA controllers.</p>
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  <p class="mainbody"><strong>Application</strong><br />
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  we utilized the  platform to improve performance of two modular genetic devices,<a href="https://2011.igem.org/Team:Peking_R/Project/Application/AG"> <u>AND gate</u></a> and<u> <a href="https://2011.igem.org/Team:Peking_R/Project/Application/BS">bistable switch</a></u><a href="https://2011.igem.org/Team:Peking_R/Project/Application/">.</a></p>
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  <p class="mainbody">&nbsp;</p>
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  <p class="mainbody"><a href="https://2011.igem.org/Team:Peking_R/Project/Application/AG">AND gate</a></p>
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      <td>&nbsp;</td>
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      <td><p class="picturemark">Figure 1. AND gate performance regulated by  different concentration of thiamine pyrophosphate (TPP). The on/off ratio of  AND gate increases with ligand concentration, while the single induction of  arabinose is diminished, resulting in a well performed AND gate.</p></td>
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  <p>&nbsp;</p>
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  <p><a href="https://2011.igem.org/Team:Peking_R/Project/Application/BS">Bistable switch</a></p>
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      <th scope="col"><img src="https://static.igem.org/mediawiki/2011/5/51/PEKINGr_Mt2.jpg" width="500" height="126" /></th>
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      <td>&nbsp;</td>
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      <td><p class="picturemark">Figure 2<strong>.</strong>  Fluorescence images of <em>E.coli</em> DH5α strain  populations with different plasmids from bistable switch mutant library. Each  plasmid contains different ribosome binding sites (RBSs) which control the  expression of <em>cI434 </em>gene, demonstrating  that the ratiometric of green cells to red cells is correlated with translation  strength. </p></td>
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  <p class="mainbody"> <u><a href="https://2011.igem.org/Team:Peking_R/Project/Application/VIO">violacein biosynthetic pathway</a></u></p>
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  <p class="mainbody">We further applied this platform to  optimize a segment of<u><a href="https://2011.igem.org/Team:Peking_R/Project/Application/VIO">violacein biosynthetic pathway</a></u>, and achieved  producing purer desired products. </p>
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      <td><p>Figure 3.  <em>E. coli</em> producing pigments. When induced by arabinose, the engineered <em>E. coli </em>produced dark-green pigments.  Upon addition of different concentration of thiamine pyrophosphate (TPP), the  color of the bacteria gradually shifted from dark-green to dark-brown.</p></td>
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   <p>&nbsp;</p>
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    <p class="aca"><strong>Parts Sandbox</strong></p>
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     <p><strong>1. <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598003">BBa_K598003</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598004">BBa_K598004</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598004"></a></p>
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    <p><em>TPP series ribocontroller</em></p>
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    <p>&nbsp;</p>
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    <p><strong>2. <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598005">BBa_K598005</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598006">BBa_K598006</a></strong></p>
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    <p><em>Theophyline series ribocontroller</em></p>
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    <p>&nbsp;</p>
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    <p><strong>3.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598007">BBa_K598007</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598008">BBa_K598008</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598008"></a></p>
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    <p><em>Ribocontroller with GFP as reporter</em></p>
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    <p>&nbsp;</p>
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    <p><strong>4.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598009">BBa_K598009</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598010">BBa_K598010</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598011">BBa_K598011</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598012">BBa_K598012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598013">BBa_K598013</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598014">BBa_K598014</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598014"></a></p>
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    <p><em>Complete plasmid contained pBAD promoter, ribocontroller and GFP</em></p>
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    <p>&nbsp;</p>
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    <p><strong>5.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598015">BBa_K598015</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598016">BBa_K598016</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598016"></a></p>
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    <p><em>Complete plasmid contained T7 promoter, ribocontroller and GFP</em></p>
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    <p>&nbsp;</p>
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    <p><strong>6.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598017">BBa_K598017</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598018">BBa_K598018</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598018"></a></p>
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    <p><em>New screened ribocontroller and the screening device</em></p>
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    <p>&nbsp;</p>
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    <p><strong>7.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598019">BBa_K598019</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598020">BBa_K598020</a></strong></p>
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    <p><em>Vio operon under ribocontroller regulation</em></p>
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    <p>&nbsp;</p>
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    <p><strong>8.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598022">BBa_K598022</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598023">BBa_K598023</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598024">BBa_K598024</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598025">BBa_K598025</a></strong><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598025"></a></p>
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    <p><em>Bistable switch device under ribocontroller regulation</em></p>
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    <p>&nbsp;</p>
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    <p><strong>9.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598026">BBa_K598026</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K598027">BBa_K598027</a></strong></p>
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    <p><em>AND gate device under ribocontroller regulation</em></p>
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<p class="mainbody"><span class="Reference">Reference:</span><a name="r101" id="r101"></a><a name="r102" id="r102"></a><a name="r103" id="r103"></a><a name="r201" id="r201"></a><a name="r202" id="r202"></a><a name="r203" id="r203"></a><a name="r204" id="r204"></a><a name="r301" id="r301"></a><a name="r302" id="r302"></a><a name="r303" id="r303"></a><a name="r304" id="r304"></a></p>
 
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<p>[1]  <a href="http://en.wikipedia.org/wiki/Hard_coding">http://en.wikipedia.org/wiki/Hard_coding</a><br />
 
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  [2] <a href="http://en.wikipedia.org/wiki/Softcoding">http://en.wikipedia.org/wiki/Softcoding</a></p>
 
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<p><span class="mainbody">[3].Gardner, Timothy S. <em>et. al.</em> (2000). Construction of a genetic toggleswitch in <em>Escherichia coli</em>. Nature 403, 339-342</span> [4].Elowitz, Michael B. and  Leibler, Stanislas (2000). A synthetic oscillatory network of transcriptional  regulators. Nature 403, 335-338  [5].Breaker, Ronald R (2004).Natural and engineered nucleicacids as  tools to explore biology. Nature 432, 838-845<br />
 
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  [6].Salis, Howard M <em>et.al.</em> (2009). Automated design of synthetic ribosome binding sitesto control protein  expression. Nat. Biotech. 27, 946-950</p>
 
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Revision as of 23:03, 5 October 2011

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Parts of iGEM 2011 Peking_R

Favorite Parts

1.BBa_K598000

TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015

This part is TPP up-regulated hammerhead ribozyme, consisting of 131 nucleotides.

 

2.BBa_K598001

Theophyline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator

This is a GFP generator regulated by theophylline responsive riboswitch. mRNA with a theophylline riboswitch in it responds to theophylline concerntration, producing different fluorescence strength of GFP. Regulated by different promoters, it would be used to demonstrate theophylline responsive curve, providing data for modeling.

 

3.BBa_K598002

Bistable Switch Mutant 68

This part is one of the mutation libraries of bistable switch modifying the ribosome binding site (RBS) of cI434 gene.

 

Parts Sandbox

1. BBa_K598003, BBa_K598004

TPP series ribocontroller

 

2. BBa_K598005, BBa_K598006

Theophyline series ribocontroller

 

3.BBa_K598007, BBa_K598008

Ribocontroller with GFP as reporter

 

4.BBa_K598009,BBa_K598010,BBa_K598011,BBa_K598012,BBa_K598013,BBa_K598014

Complete plasmid contained pBAD promoter, ribocontroller and GFP

 

5.BBa_K598015,BBa_K598016

Complete plasmid contained T7 promoter, ribocontroller and GFP

 

6.BBa_K598017,BBa_K598018

New screened ribocontroller and the screening device

 

7.BBa_K598019,BBa_K598020

Vio operon under ribocontroller regulation

 

8.BBa_K598022,BBa_K598023,BBa_K598024,BBa_K598025

Bistable switch device under ribocontroller regulation

 

9.BBa_K598026,BBa_K598027

AND gate device under ribocontroller regulation

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