Difference between revisions of "Loop Assembly"

Revision as of 20:56, 29 November 2018

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 as "fusion sites." Samples of basic parts (Promoter, RBS, CDS, etc) are flanked by defined fusion sites which are dictated by the part type and/or desired position during assembly. When part samples are cut with a Type IIS enzyme will produce compatible overhangs at the fusion sites which will allow the parts to be ligated in the desired order. This means that a single one-pot assembly reaction can include multiple basic parts (Promoter, RBS, CDS, Terminator) and result in a complete transcriptional unit (composite part).

See the diagram below for an example of a Type IIS assembly reaction.

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

Level 0 (basic parts)

For basic parts

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 (composite parts), and Level 2 as multi-transcriptional unit assemblies.

PhytoBricks info goes here

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 Type IIS assembly methods, Loop is structured to allow for increasing complexity of assemblies (Level 1, Level 2). However, the recursive design in Loop allows for the same two cloning vectors to be used to create Level 3 and Level 4.

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

@@ Line 24: / Line 24: @@
 ==Background==
-Type IIS assembly methods use Type IIS restriction enzymes, which cleave DNA outside their recognition site. First introduced in Golden Gate cloning ([https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647 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.
+Type IIS assembly methods use Type IIS restriction enzymes, which cleave DNA outside their recognition site. First introduced in Golden Gate cloning ([https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647 Engler et al. 2008]), these assembly methods standardize the cleavage sites of the Type IIS enzymes as "fusion sites." Samples of basic parts (Promoter, RBS, CDS, etc) are flanked by defined fusion sites which are dictated by the part type and/or desired position during assembly. When part samples are cut with a Type IIS enzyme will produce compatible overhangs at the fusion sites which will allow the parts to be ligated in the desired order. This means that a single one-pot assembly reaction can include multiple basic parts (Promoter, RBS, CDS, Terminator) and result in a complete transcriptional unit (composite part).
+See the diagram below for an example of a Type IIS assembly reaction.
 [[File:Assembly_-_GoldenGate_Example.jpeg|thumb|center|Example of Type IIS enzyme (BsaI) cleaving multiple parts for assembly (Fig 1 from Patron et al. 2015)]]
@@ Line 50: / Line 52: @@
 ===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.
+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.
@@ Line 58: / Line 60: @@
 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 ...
+Like other Type IIS assembly methods, Loop is structured to allow for increasing complexity of assemblies (Level 1, Level 2). However, the recursive design in Loop allows for the same two cloning vectors to be used to create Level 3 and Level 4.