Team:Cambridge-JIC/Marchantia/RFC

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Cambridge iGEM 2014


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.


  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

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
Figure 1: Illustration of the idea behind mӧsbi's modularity

Rise of the Titles

Third part here