Difference between revisions of "Team:Freiburg/Parts"

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Bitte auf Artnamen achten (Vorname Groß, Nachname klein, alles kursiv (Philipp)) Bezüglich pop: warum ist pSB1C3 ein doofes Backbone für die expression? warum wird iGEM viel mehr proteinexpression machen (in-vitro ist en vogue)? Warum habt ihr plötzlich angefangen ein anderes Backbone genutzt? (Resistenz, lacI etc). Kurz: Warum ist pOP cool?
 
Bitte auf Artnamen achten (Vorname Groß, Nachname klein, alles kursiv (Philipp)) Bezüglich pop: warum ist pSB1C3 ein doofes Backbone für die expression? warum wird iGEM viel mehr proteinexpression machen (in-vitro ist en vogue)? Warum habt ihr plötzlich angefangen ein anderes Backbone genutzt? (Resistenz, lacI etc). Kurz: Warum ist pOP cool?
 
</br> neuer Versuch (9.9.15 Lara)
 
</br> neuer Versuch (9.9.15 Lara)
 +
</br> Ramona: Hab nochmal etwas umgebaut. Denke, der pOP kommt jetzt besser raus, die Details dazu findet man aber nach wie vor auf seiner eigenen Seite, die jetzt allerdings auch direkt im Text verlinkt ist.
 
</div>
 
</div>
<p>
 
Since our project involved expression of many antigenic peptides we decided to share those antigen-sequences with the iGEM community. We obtained most of the sequences via paper research and we would like to give special thanks to the group of Prof. Dr. Michael Hust (TU Braunschweig) who provided us with expression plasmids for the <em>Salmonella</em> Typhimurium antigen and a corresponding single chain variable fragment. The codon optimization tool from Integrated DNA Technologies was used to improve most sequences for expression in <em>E. coli</em>. We also removed all restriction sites that are not allowed in RFC[10], so that all sequences are compatible with the submission vector pSB1C3.
 
When we started working with our first plasmids for this project, we decided to use a specific nomenclature. Every plasmid name starts with “pIG15” which is short for “plasmid igem 2015”.
 
According to this, we named the plasmids containing our biobricks in the shipping backbone pRIG15 (“pRIG” as in “brick”)
 
  
 +
<p>
 +
Our project involved the expression of many antigenic peptides. Their immunodominant properties make them feasible for many medical purposes. As ‘Health and Medicine’ is one of the most popular tracks chosen by iGEM teams, we want to share the sequences encoding for these peptides with the iGEM community. Thus, future iGEM teams have the opportunity to take advantage from our research if they are planning to work in the field of diagnostics.
 
</br>
 
</br>
 +
Most of the sequences were obtained by paper research and have been synthesized by Integrated DNA technologies, engaging their special offer for iGEM teams. Before ordering, we used the IDT codon optimization tool to optimize the sequences for efficient expression in <i>E. coli</i>. Planning to submit the sequences to the registry, recognition sites for restriction enzymes used for standard cloning have been removed without causing amino acid changes.
 
</br>
 
</br>
In the table below we listed all our biobricks and our biobrick improvement, pOP. We decided on pOP as our favorite biobrick because it provides the iGEM communitiy with an iGEM conform backbone for protein expression. When we started our project we faced the problem of pSB1C3 being a plasmid designated for cloning colliding with our need of a plasmid for expression of proteins.  
+
The group of Prof. Dr. Michael Hust (TU Braunschweig) provided us with an <a href="http://parts.igem.org/Part:BBa_K1621006">antigen specific for <i>Salmonella</i> Typhimurium</a> and a <a href="http://parts.igem.org/Part:BBa_K1621007">corresponding single chain variable fragment (scFv)</a>. They generated the scFv themselves after identifying the immunodominant properties of the protein. Thanks to their work we are able to provide the registry with a specific pair of antigen and scFv.
 
</br>
 
</br>
 
+
All our biobricks are summarized in the table below. Additionally, the table guides you to more detailed info pages as well as to the corresponding registry pages.
We think that expression of proteins is a rather big topic for future iGEM teams, since working <em>in-vitro</em> is an upcoming issue of synthetic biology. (Beispiele? kommt noch)
+
Expression of proteins is usually done in specialized expression strains, that in the case of <em>E. coli</em> often carry a resistence against chloramphenicol. That is why plasmids used for expression should have an additional resistance making selection of positively transformed clones possible. The iGEM standard backbone pSB1C3 only contains a chloramphenicol resistance making it difficult to use in combination with expression strains like <em>E. coli</em> BL21 or Rosetta.
+
Since protein expression sometimes has negative consequences for the cell expressing the protein it is useful to have a tool, which allows induction of protein expression at a certain timepoint. One system which is often used is a lac inducible promoter system, that can be started with IPTG. pSB1C3 does not have any default promotor inserted at all, making protein expression more difficult because an appropriate promotor has to be inserted every time.
+
 
</br>
 
</br>
One additional disadvantage of pSB1C3 is the high copy ori which is really useful for cloning, but disturbs protein expression.
 
Therefore an iGEM backbone adapted for protein expression seemed to be a quite good idea. We improved the pSB1C3 standard vector in a way that it can be used to express proteins in <em>E. coli</em> but still exhibits all the properties of an iGEM standard vector.
 
</br>
 
Details on which parts of pSB1C3 we changed and how we did this can be found <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pOP-vector" title="pop_-_protein_expression_meets_igem_standards">here</a>.
 
 
</br>
 
</br>
 +
Although these parts significantly expand the registry in terms of diagnostics, none of them is our favorite biobrick. To reach the high yields of protein expression we needed for our project, we used a commercial vector optimized for inducible protein overexpression. This was unavoidable because inserting the required parts (like the lacI gene or a lacI repressible promoter) into pSB1C3 did not result in satisfying protein amounts. </br>
 +
For improving a BioBrick, we developed a plasmid backbone that is adapted for efficient protein overexpression while fitting to iGEM standards. We wanted to enable every future iGEM team to perform highly efficient protein overexpression based on this backbone. Since working <i>in vitro</i> is an upcoming issue in synthetic biology, this is at once our favorite biobrick. <a href="">Find out more about the pOP vector </a>and see if it fits also your requirements!
 +
</p>
  
 
</p>
 
 
<div class="flexbox">
 
<div class="flexbox">
 
<p>
 
<p>
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</tr>
 
</tr>
 
<tr class="row1">
 
<tr class="row1">
<td class="col1"> <a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621000" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621000">BBa_K1621000</a></td><td class="col0"> Rubella Virus specific antigenic epitopes derived from glycoprotein E1</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_6" title="pRIG15_6">pRIG15_6</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621009" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621009">BBa_K1621009</a></td><td class="col0"> Standardized plasmid backbone optimized for protein overexpression</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pOP-vector" title="pop_-_protein_expression_meets_igem_standards">pOP</a></td>
 
</tr>
 
</tr>
 
<tr class="row2">
 
<tr class="row2">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621001" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621001">BBa_K1621001</a></td><td class="col0"> Varicella Zoster Virus specific antigenic epitopes derived from glycoprotein E</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_7" title="prig15_7">pRIG15_7</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621007" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621007">BBa_K1621007</a></td><td class="col0"> scFv binding specifically to the <i>Salmonella</i> Typhimurium derived antigen</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_13" title="prig15_13">pRIG15_13</a></td>
 
</tr>
 
</tr>
 
<tr class="row3">
 
<tr class="row3">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621002" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621002">BBA_K1621002</a></td><td class="col0"> Herpes Simplex specific antigenic epitopes derived from glycoprotein G</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_8" title="prig15_8">pRIG15_8</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621006" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621006">BBa_K1621006</a></td><td class="col0"><i> Salmonella</i> Typhimurium specific antigenic protein (DHAD)<td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_15" title="prig15_15">pRIG15_15</a></td>
 
</tr>
 
</tr>
 
<tr class="row4">
 
<tr class="row4">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621003" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621003">BBa_K1621003</a></td><td class="col0"> <i> Clostridium tetani</i> specific antigenic epitopes derived from tetanus neurotoxin (TeNT_Hc)</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_11" title="prig15_11">pRIG15_11</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621005" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621005">BBa_K1621005</a></td><td class="col0"> <i> Treponema pallidum</i> specific antigenic peptides derived from bacterioferritin (TpF1)</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_18" title="prig15_18">pRIG15_18</a></td>
 
</tr>
 
</tr>
 
<tr class="row5">
 
<tr class="row5">
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</tr>
 
</tr>
 
<tr class="row6">
 
<tr class="row6">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621005" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621005">BBa_K1621005</a></td><td class="col0"> <i> Treponema pallidum</i> specific antigenic peptides derived from bacterioferritin (TpF1)</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_18" title="prig15_18">pRIG15_18</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621003" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621003">BBa_K1621003</a></td><td class="col0"> <i> Clostridium tetani</i> specific antigenic epitopes derived from tetanus neurotoxin (TeNT_Hc)</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_11" title="prig15_11">pRIG15_11</a></td>
 
</tr>
 
</tr>
 
<tr class="row7">
 
<tr class="row7">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621006" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621006">BBa_K1621006</a></td><td class="col0"><i> Salmonella</i> Typhimurium specific antigenic protein (DHAD)<td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_15" title="prig15_15">pRIG15_15</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621002" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621002">BBA_K1621002</a></td><td class="col0"> Herpes Simplex specific antigenic epitopes derived from glycoprotein G</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_8" title="prig15_8">pRIG15_8</a></td>
 
</tr>
 
</tr>
 
<tr class="row8">
 
<tr class="row8">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621007" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621007">BBa_K1621007</a></td><td class="col0"> scFv binding specifically to the <i>Salmonella</i> Typhimurium derived antigen</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_13" title="prig15_13">pRIG15_13</a></td>
+
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621001" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621001">BBa_K1621001</a></td><td class="col0"> Varicella Zoster Virus specific antigenic epitopes derived from glycoprotein E</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_7" title="prig15_7">pRIG15_7</a></td>
 
</tr>
 
</tr>
 
<tr class="row9">
 
<tr class="row9">
<td class="col1"><a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621009" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621009">BBa_K1621009</a></td><td class="col0"> Standardized plasmid backbone optimized for protein overexpression</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pOP-vector" title="pop_-_protein_expression_meets_igem_standards">pOP</a></td>
+
<td class="col1"> <a class="urlextern" href="http://parts.igem.org/Part:BBa_K1621000" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_K1621000">BBa_K1621000</a></td><td class="col0"> Rubella Virus specific antigenic epitopes derived from glycoprotein E1</td><td class="col0"><a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/pRIG15_6" title="pRIG15_6">pRIG15_6</a></td>
 
</tr>
 
</tr>
 +
 +
 +
 +
 +
 +
 
</table></div>
 
</table></div>
  

Revision as of 19:45, 10 September 2015

""

Biobricks

Hier fehlt auf jeden Fall noch die Nennung unseres Favorite Biobricks mit erklärung warum das so ist. Ich denke in unserem Fall wird das der pOP sein oder? (Stefan)
Bitte auf Artnamen achten (Vorname Groß, Nachname klein, alles kursiv (Philipp)) Bezüglich pop: warum ist pSB1C3 ein doofes Backbone für die expression? warum wird iGEM viel mehr proteinexpression machen (in-vitro ist en vogue)? Warum habt ihr plötzlich angefangen ein anderes Backbone genutzt? (Resistenz, lacI etc). Kurz: Warum ist pOP cool?
neuer Versuch (9.9.15 Lara)
Ramona: Hab nochmal etwas umgebaut. Denke, der pOP kommt jetzt besser raus, die Details dazu findet man aber nach wie vor auf seiner eigenen Seite, die jetzt allerdings auch direkt im Text verlinkt ist.

Our project involved the expression of many antigenic peptides. Their immunodominant properties make them feasible for many medical purposes. As ‘Health and Medicine’ is one of the most popular tracks chosen by iGEM teams, we want to share the sequences encoding for these peptides with the iGEM community. Thus, future iGEM teams have the opportunity to take advantage from our research if they are planning to work in the field of diagnostics.
Most of the sequences were obtained by paper research and have been synthesized by Integrated DNA technologies, engaging their special offer for iGEM teams. Before ordering, we used the IDT codon optimization tool to optimize the sequences for efficient expression in E. coli. Planning to submit the sequences to the registry, recognition sites for restriction enzymes used for standard cloning have been removed without causing amino acid changes.
The group of Prof. Dr. Michael Hust (TU Braunschweig) provided us with an antigen specific for Salmonella Typhimurium and a corresponding single chain variable fragment (scFv). They generated the scFv themselves after identifying the immunodominant properties of the protein. Thanks to their work we are able to provide the registry with a specific pair of antigen and scFv.
All our biobricks are summarized in the table below. Additionally, the table guides you to more detailed info pages as well as to the corresponding registry pages.

Although these parts significantly expand the registry in terms of diagnostics, none of them is our favorite biobrick. To reach the high yields of protein expression we needed for our project, we used a commercial vector optimized for inducible protein overexpression. This was unavoidable because inserting the required parts (like the lacI gene or a lacI repressible promoter) into pSB1C3 did not result in satisfying protein amounts.
For improving a BioBrick, we developed a plasmid backbone that is adapted for efficient protein overexpression while fitting to iGEM standards. We wanted to enable every future iGEM team to perform highly efficient protein overexpression based on this backbone. Since working in vitro is an upcoming issue in synthetic biology, this is at once our favorite biobrick. Find out more about the pOP vector and see if it fits also your requirements!

biobrick short description detailed desription
BBa_K1621009 Standardized plasmid backbone optimized for protein overexpressionpOP
BBa_K1621007 scFv binding specifically to the Salmonella Typhimurium derived antigenpRIG15_13
BBa_K1621006 Salmonella Typhimurium specific antigenic protein (DHAD)pRIG15_15
BBa_K1621005 Treponema pallidum specific antigenic peptides derived from bacterioferritin (TpF1)pRIG15_18
BBa_K1621004 Human Immunodeficiency Virus specific antigenic epitopes derived from a polyprotein called gag/tat/pol/envpRIG15_17
BBa_K1621003 Clostridium tetani specific antigenic epitopes derived from tetanus neurotoxin (TeNT_Hc)pRIG15_11
BBA_K1621002 Herpes Simplex specific antigenic epitopes derived from glycoprotein GpRIG15_8
BBa_K1621001 Varicella Zoster Virus specific antigenic epitopes derived from glycoprotein EpRIG15_7
BBa_K1621000 Rubella Virus specific antigenic epitopes derived from glycoprotein E1pRIG15_6