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.
A Type IIS assembly method uses Type IIS restriction enzymes, which are offsite cutters; they cut DNA at a specific distance from their recognition site. This allows for specified overhangs left by the Type IIS enzymes.
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
Assembly Compatibility / Illegal Sites
iGEM Type IIS compatible parts must not contain the Bsa1 and Sap1 Type IIS recognition sequences, as these restriction enzymes are required for assembly. Parts that have these “illegal” recognition sites are not compatible with the standard.
| Enzyme | Type | Sequence |
|---|---|---|
| BsaI | Illegal | GGTCTC |
| SapI | Illegal | GCTCTTC |
Fusion Sites
Below are the fusion sites for Level 0 (Basic Parts) when cut with Bsa1.
| Fusion Site 5' | Part Type | Fusion Site 3' |
|---|---|---|
| GGAG | Promoter | TACT |
| TACT | 5'UTR | AATG |
| AATG | CDS | GCTT |
| GCTT | Terminator | 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. These fusion sites have also been tested 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).
Prefix and Suffix
| Prefix | Part Type | Suffix |
|---|---|---|
|
5' GGTCTC N GGAG CCAGAG N CCTC |
Promoter Promoter |
TACT N GAGACC 3' ATGA N CTCTGG |
|
5' GGTCTC N TACT CCAGAG N ATGA |
5'UTR 5'UTR |
AATG N GAGACC 3' TTAC N CTCTGG |
|
5' GGTCTC N AATG CCAGAG N TTAC |
CDS CDS |
GCTT N GAGACC 3' CGAA N CTCTGG |
|
5' GGTCTC N GCTT CCAGAG N CGAA |
Terminator Terminator |
CGCT N GAGACC 3' GCGA N CTCTGG |
Accepted Standards: BioBrick RFC[10] | iGEM Type IIS RFC[1000]