Team:Cambridge-JIC/Marchantia/RFC

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<div> I will put in divisions to hold the pieces of information we have specified in the WIKI spreadsheet</div>
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<div align="center"><a href="https://2014.igem.org/wiki/index.php?title=Team:Cambridge-JIC/Marchantia/RFC&action=edit"> Edit this page </a></div>
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                        <h1>Request for Comments</h1>
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                        <font color="black" style="BACKGROUND-COLOR: #E6E6E6">MoClo as a standard assembly in plants</font>
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    <div class="content-section-a" id="Overview">
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                    <h2 class="section-heading">BBF RFC 105: Standardized Plant Syntax</h2>
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                    <div>
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<h4>Purpose</h4>
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<p>
 +
The difference in the biology of plants unveils a need for new standards. We decided to write a Request for Comments (RFC) to unite the plant SynBio community by defining a common language for plant synthetic biology, extensible to all other Eukaryotes. Its syntax is based on the Golden Gate System<sup>1</sup> cloning method which uses TypeIIS restriction enzymes; variations, such as GB2.0<sup>2</sup> and MoClo<sup>3</sup>, are already being used across the field. This RFC brings together their common features and sets a consensus across the plant field for construct assembly and part repositories.
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</p>
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<p>
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Marchantia polymorpha, a primitive liverwort, is being established as a new model organism for plant Synthetic Biology<sup>4</sup>; Contrary to historical model species, the choice of M. polymorpha has preceded its establishment. This is an opportunity to set standards in a coherent and united way paving the way for faster and more efficient sharing of information and parts.
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</p>
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<p>
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The fewer the rules defining a standard, the more widely that standard is adopted. The introduction of this new chassis with its novel tool kit is the occasion to allow more flexibility in the Registry<sup>5</sup>, opening it to wider usage and contributions.
 +
</p>
 +
<p>
 +
Thank you to the UEA and Valencia iGEM teams for their collaborations.
 +
</p>
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<br>
 +
 
 +
 
 +
<h3> Table of Contents </h3>
 +
 
 +
<UL>
 +
BBF RFC 105: Standardized Plant Syntax <br>
 +
1. Purpose <br>
 +
2. Relation to other BBF RFCs <br>
 +
3. Copyright Notice <br>
 +
4. A Common Type IIs Grammar for Eukaryotic transcriptional units <br>
 +
<ul>
 +
i. Introduction to the Golden Gate Standards <br>
 +
ii. Part Standards <br>
 +
iii. Common Vector for Level 0 Parts <br>
 +
iv. Domestication Protocol of Level 0 Parts <br></ul>
 +
5. Golden Gate Assembly Rules <br>
 +
6. Multigenic constructs <br>
 +
a. GoldenBraid assembly <br>
 +
b. MoClo assembly <br>
 +
7. Unified features of binary vector for agrobacterium-mediated delivery <br><ul>
 +
a. Introduction to Agrobacterium-mediated delivery <br>
 +
b. Standard Features for Agrobacterium-mediated delivery backbones <br></ul>
 +
8. Applications <br><ul>
 +
a. Library construction <br>
 +
b. Expression Cassettes <br></ul>
 +
9. Marchantia polymorpha, a model organism <br><ul>
 +
i. Introduction to the new chassis <br>
 +
ii. Marchantia OpenParts Library and Codon optimization <br>
 +
iii. Marchantia Agoro-mediated Transformation protocol and T-plasmid specification <br></ul>
 +
10. Nicotiana benthamiana and Arabidopsis thaliana: model dicotyledenous plants <br><ul>
 +
i. Description of the chassis <br>
 +
ii. GoldenGate parts and codon-optimisation for model dicotyledenous plants <br>
 +
iii. Agrobacterium-mediated Transformation protocol <br></ul>
 +
11. Additional Resources and Information <br>
 +
12. Authors’ Contact Information <br>
 +
13. References <br>
 +
 
 +
</UL>
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<div class="container">
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        <div class="col-lg-9 col-sm-push-0.75  col-sm-6">
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            <hr class="section-heading-spacer">
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            <div class="clearfix"></div>
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            <div>
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<h4>Standardized Fusion Sequences</h4>
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<p>One part of the RFC was reaching a consensus on fusion sequences:</p>
 +
<table>
 +
<tr>
 +
<th>
 +
Nature of Simplest Part
 +
</th>
 +
<th>
 +
5′ 4bp overhang
 +
</th>
 +
<th>
 +
3′ 4bp overhang
 +
</th>
 +
</tr>
 +
<tr>
 +
<td>
 +
Pro = Promoter
 +
</td>
 +
<td>
 +
GGAG
 +
</td>
 +
<td>
 +
TACT
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
5U = 5′ UTR up to atg
 +
</td>
 +
<td>
 +
TACT
 +
</td>
 +
<td>
 +
AATG
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
5U(f) = 5′ UTR (for fusing to NT1 modules)
 +
</td>
 +
<td>
 +
TACT
 +
</td>
 +
<td>
 +
CCAT
 +
</td>
 +
</tr>
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<tr>
 +
<td>
 +
NT1 = N-terminal tag/signal peptide
 +
</td>
 +
<td>
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CCAT
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</td>
 +
<td>
 +
AATG
 +
</td>
 +
</tr>
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<tr>
 +
<td>
 +
NT2 =  N-terminal tag/signal peptide
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</td>
 +
<td>
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AATG
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</td>
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<td>
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AGCC
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</td>
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</tr>
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<tr>
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<td>
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CDS1 = Coding sequence
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</td>
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<td>
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AATG
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</td>
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<td>
 +
GCTT
 +
</td>
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</tr>
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<tr>
 +
<td>
 +
CDS2 = Coding sequence (for fusing with SP)
 +
</td>
 +
<td>
 +
AGCC
 +
</td>
 +
<td>
 +
GCTT
 +
</td>
 +
</tr>
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<tr>
 +
<td>
 +
CDS1ns = Coding sequence (no stop) (for fusing to CT modules)
 +
</td>
 +
<td>
 +
AATG
 +
</td>
 +
<td>
 +
TTCG
 +
</td>
 +
</tr>
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<tr>
 +
<td>
 +
CDS2ns = Coding sequence (no stop) (for fusing with SP and CT modules)
 +
</td>
 +
<td>
 +
AGCC
 +
</td>
 +
<td>
 +
TTCG
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
CT = C-terminal tag/fusion protein
 +
</td>
 +
<td>
 +
TTCG
 +
</td>
 +
<td>
 +
GCTT
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
3U= 3′ UTR
 +
</td>
 +
<td>
 +
GCTT
 +
</td>
 +
<td>
 +
GGTA
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
Ter= terminator
 +
</td>
 +
<td>
 +
GGTA
 +
</td>
 +
<td>
 +
CGCT
 +
</td>
 +
</tr>
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<tr>
 +
<td>
 +
OP1
 +
</td>
 +
<td>
 +
GGAG
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</td>
 +
<td>
 +
TGAC
 +
</td>
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</tr>
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<tr>
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<td>
 +
OP2
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</td>
 +
<td>
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TGAC
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</td>
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<td>
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TCCC
 +
</td>
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</tr>
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<tr>
 +
<td>
 +
Mini Pro + 5U
 +
</td>
 +
<td>
 +
TCCC
 +
</td>
 +
<td>
 +
ATG
 +
</td>
 +
</tr>
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</table>
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<br><br>
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<figcaption> Standardized Fusion Table</figcaption>
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/4/4f/Standard_Table.png"width=700px">
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<img src="https://static.igem.org/mediawiki/2014/8/84/Cambridge-JIC_Framework_overview.png" width="700px">
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<figcaption>Figure 1: Illustration of the idea behind mӧsbi's modularity</figcaption>
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</figure>-->
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<p>Click <a href="https://static.igem.org/mediawiki/2014/6/61/Cambridge-JIC_RFC105%286%29DO2_%281%29.pdf">here</a> to download the full RFC as it stands on the 17th of October.</p>
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<ol class="reference">
 +
<li> Engler C, Youles M, Gruetzner R, Ehnert T-M, Werner S, et al. (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol: doi:10.1021/sb4001504. </li>
 +
<li> Sarrion-Perdigones A., Falconi E.E., Zandalinas S.I., Juarez P., Fernandez-del-Carmen A., Granell A., Orzaez D. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 2011;6:e21622. </li>
 +
<li> Weber E., Engler C., Gruetzner R., Werner S., Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One 2011;6:e16765. </li>
 +
<li> <a href="http://synbio.org.uk/marchantia/"> http://synbio.org.uk/marchantia/ </a></li>
 +
<li> <a href="http://parts.igem.org/Main_Page"> http://parts.igem.org/Main_Page </a></li>
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</ol>
</body>
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Latest revision as of 22:53, 17 October 2014

Cambridge iGEM 2014

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Request for Comments

MoClo as a standard assembly in plants

BBF RFC 105: Standardized Plant Syntax

Purpose

The difference in the biology of plants unveils a need for new standards. We decided to write a Request for Comments (RFC) to unite the plant SynBio community by defining a common language for plant synthetic biology, extensible to all other Eukaryotes. Its syntax is based on the Golden Gate System1 cloning method which uses TypeIIS restriction enzymes; variations, such as GB2.02 and MoClo3, are already being used across the field. This RFC brings together their common features and sets a consensus across the plant field for construct assembly and part repositories.

Marchantia polymorpha, a primitive liverwort, is being established as a new model organism for plant Synthetic Biology4; Contrary to historical model species, the choice of M. polymorpha has preceded its establishment. This is an opportunity to set standards in a coherent and united way paving the way for faster and more efficient sharing of information and parts.

The fewer the rules defining a standard, the more widely that standard is adopted. The introduction of this new chassis with its novel tool kit is the occasion to allow more flexibility in the Registry5, opening it to wider usage and contributions.

Thank you to the UEA and Valencia iGEM teams for their collaborations.


Table of Contents

    BBF RFC 105: Standardized Plant Syntax
    1. Purpose
    2. Relation to other BBF RFCs
    3. Copyright Notice
    4. A Common Type IIs Grammar for Eukaryotic transcriptional units
      i. Introduction to the Golden Gate Standards
      ii. Part Standards
      iii. Common Vector for Level 0 Parts
      iv. Domestication Protocol of Level 0 Parts
    5. Golden Gate Assembly Rules
    6. Multigenic constructs
    a. GoldenBraid assembly
    b. MoClo assembly
    7. Unified features of binary vector for agrobacterium-mediated delivery
      a. Introduction to Agrobacterium-mediated delivery
      b. Standard Features for Agrobacterium-mediated delivery backbones
    8. Applications
      a. Library construction
      b. Expression Cassettes
    9. Marchantia polymorpha, a model organism
      i. Introduction to the new chassis
      ii. Marchantia OpenParts Library and Codon optimization
      iii. Marchantia Agoro-mediated Transformation protocol and T-plasmid specification
    10. Nicotiana benthamiana and Arabidopsis thaliana: model dicotyledenous plants
      i. Description of the chassis
      ii. GoldenGate parts and codon-optimisation for model dicotyledenous plants
      iii. Agrobacterium-mediated Transformation protocol
    11. Additional Resources and Information
    12. Authors’ Contact Information
    13. References

Standardized Fusion Sequences

One part of the RFC was reaching a consensus on fusion sequences:

Nature of Simplest Part 5′ 4bp overhang 3′ 4bp overhang
Pro = Promoter GGAG TACT
5U = 5′ UTR up to atg TACT AATG
5U(f) = 5′ UTR (for fusing to NT1 modules) TACT CCAT
NT1 = N-terminal tag/signal peptide CCAT AATG
NT2 = N-terminal tag/signal peptide AATG AGCC
CDS1 = Coding sequence AATG GCTT
CDS2 = Coding sequence (for fusing with SP) AGCC GCTT
CDS1ns = Coding sequence (no stop) (for fusing to CT modules) AATG TTCG
CDS2ns = Coding sequence (no stop) (for fusing with SP and CT modules) AGCC TTCG
CT = C-terminal tag/fusion protein TTCG GCTT
3U= 3′ UTR GCTT GGTA
Ter= terminator GGTA CGCT
OP1 GGAG TGAC
OP2 TGAC TCCC
Mini Pro + 5U TCCC ATG


Standardized Fusion Table

Click here to download the full RFC as it stands on the 17th of October.

  1. Engler C, Youles M, Gruetzner R, Ehnert T-M, Werner S, et al. (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol: doi:10.1021/sb4001504.
  2. Sarrion-Perdigones A., Falconi E.E., Zandalinas S.I., Juarez P., Fernandez-del-Carmen A., Granell A., Orzaez D. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 2011;6:e21622.
  3. Weber E., Engler C., Gruetzner R., Werner S., Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One 2011;6:e16765.
  4. http://synbio.org.uk/marchantia/
  5. http://parts.igem.org/Main_Page

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