Loop Assembly

Revision as of 20:26, 29 November 2018 by Vinoo (Talk | contribs)

As we prepare for iGEM 2019, I have been trying to decide how to support Type IIS assembly methods for the Registry and the 2019 iGEM Competition. Since [http://2016.igem.org/Resources/Plant_Synthetic_Biology/PhytoBricks 2016], iGEM has allowed teams to submit parts in the PhytoBricks (Type IIS Golden Gate) standard, but we have not fully supported any Type IIS standard.

Our current proposal is as follows:

iGEM fully adopts the Loop Assembly standard with Level 0 (basic) parts adhering to the MoClo and/or PhytoBricks Golden Gate assembly standards.

Adoption of Loop would include:

  • giving teams full credit for submitting samples in this standard
  • updating the Registry software to support the standard
  • creating documentation for our users
  • distributing a collection of basic parts and cloning vectors in the 2019 Distribution Kit.


iGEM would, of course, continue to support and send out BioBrick parts for the foreseeable future.



Before we finalize the plans for adopting Loop, I would like to collect input from our community. If you have any comments or thoughts on iGEM adopting and supporting Loop (including the MoClo and/or PhytoBricks assembly standards), please send an email to vinoo (at) igem (dot) org.

For more information please read below and visit the linked resources.

- Vinoo Selvarajah, Director of the Registry


Background

Type IIS assembly methods use Type IIS restriction enzymes, which cleave DNA outside their recognition site. First introduced in Golden Gate cloning (Engler et al. 2008), these assembly methods standardize the cleavage sites of the Type IIS enzymes to allow for multiple parts to be assembled in one reaction. When designed, two or more part samples cut with a Type IIS enzyme will produce compatible overhangs which will allow the parts to be ligated in the desired order. These standardized overhangs are often referred to as fusion sites.

The MoClo assembly standard (Weber et al. 2011) built upon Golden Gate and defined a standard by which parts could be assembled in increasing complexity: Level 0 as basic parts, Level 1 as transcriptional units, and Level 2 as multi-transcriptional unit assemblies.

PhytoBricks info goes here

  • Example of TypeIIS enzyme (BsaI) cleaving multiple parts for assembly (Fig 1 from Patron et al. 2015)

  • MoClo parts and fusion sites (Figure 1 from Weber et al. 2011)

  • PhytoBrick parts and fusion sites (Figure 3 from Patron et al. 2015)


Loop Assembly

Why Loop Assembly

Adoption

We know several groups, including academic/research labs and industry, that are already using Loop Assembly. We want to ensure that the assembly method we support is widely used, tested, and has flexibility for different applications and groups.

Loop Assembly is compatible with a variety of Golden Gate standards for Level 0 parts, most importantly MoClo and PhytoBricks. Both of these standards are widely used, and MoClo's fusion sites completely overlap with PhytoBricks.


Flexibility and Complexity

Loop uses two Type IIS sites for assembly: BsaI (6bp recognition site) and SapI (a 7bp recognition site). Parts designed to be used in Loop only need to be free of these two restriction sites, meaning theoretically part standardization should be easier than BioBrick RFC10 (4 illegal restriction sites).

Like other Typer IIS assembly methods, Loop is structured to ...


OpenMTA

The Loop Assembly vectors outlined in the paper are under the OpenMTA license for unrestricted sharing. iGEM/Registry can distribute and modify these vectors as needed, and so can iGEM teams. For more information on the OpenMTA license, developed by OpenPlant and The BioBricks Foundation, please read