Help:Standards/Assembly/Type IIS
Motivation & Discussion
With a Type IIS assembly method, multiple parts can be assembled easily and reliably in a single reaction, and this can be scaled further to create more complex genetic circuits quickly, efficiently, and affordably.
Currently, there are several Type IIS assembly standards in use, many of these were designed for specific purposes. iGEM believes strongly in standards, and as we approach Type IIS assembly methods we want to support one that we believe will be useful, reliable, long-lasting and widespread for the synthetic biology community.
We want to ensure that the assembly standard and method we support is widely used, tested, and has flexibility for different applications and groups.
Technical Specifications
Compatibility/Illegal Sites
iGEM Type IIS compatible parts must not contain the Bsa1 and Sap1 Type IIS restriction enzyme sites, as these are required for assembly. Parts that have these “illegal” restriction sites are not compatible with the standard.
| Enzyme | Type | Sequence |
|---|---|---|
| BsaI | Illegal | GGTCTC |
| SapI | Illegal | GCTCTTC |
Fusion Sites
| Fusion Site 5' |
Part Type | Fusion Site 3' |
| GGAG | Promoter | TACT |
| TACT | 5'UTR | AATG |
| AATG | CDS | GCTT |
| GCTT | CDS | CGCT |
Why were these fusion sites selected?
These fusion sites were outlined in the original MoClo paper (Weber, et al. 2011). They have since been adopted in other Type IIS assembly standards. As an example, these MoClo fusion sites completely overlap with the PhytoBricks standard. We have also tested these fusion sites against NEB’s 4bp ligation fidelity tool [link], finding them to be completely reliable when used as a set in a reaction.
To ensure maximum compatibility, and because of a long history of use, iGEM has adopted these fusion sites for the following basic parts. As a note, iGEM has elected to not define the fusion sites within a CDS region (AGGT, for MoClo).
Accepted Standards: BioBrick RFC[10] | iGEM Type IIS RFC[1000]