Creating Our Collection
Designed for out-of-the box modular assembly
Chlorophyll capture and degradation has various applications in the canola industry and beyond. The water-soluble chlorophyll binding protein 6GIX and the chlorophyll degradation enzymes CBR, 7-HCAR, SGR, and PPH are the first of their kind in the iGEM registry. The parts in this collection will allow for creation of genetic circuits that are ideal for high levels of protein production, secretion, and simple purification.
When designing our collection, we were interested in creating modular parts that could be used in Golden Gate assembly right out of the distribution kit, without the need to first domesticate them in a Golden Gate entry vector. As such, we purposely chose to make these parts incompatible with the iGEM Type IIS RFC assembly standard by including the BsaI restriction site and MoClo standard fusion site within the part’s submitted sequence. We believe that including these sites within the sequence will reduce the cloning steps and streamline the workflow of other iGEM teams who will use these parts in the future.
As per the MoClo assembly standard, the 5’ and 3’ fusion sequences included in the parts are outlined below (Weber et al., 2011). Including these standard sites allows the parts in this collection to be used with any MoClo-compatible or Type IIS RFC standard parts, which greatly extends the usability.
Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).
You can learn more about Golden Gate Assembly in our appendix
Our parts collection contains various basic parts as well as entire functional genetic circuits. Overall, we contributed:
- Six signal peptide parts for protein secretion
- Six novel protein-coding sequences for chlorophyll capture and enzymatic degradation
- A universal spacer and 6xHis tag for purification of electronegative proteins
- An improved RFP Golden Gate flipper device for domestication of BioBrick-compatible vectors to Golden Gate destination vectors
- A Promoter-rbs composite part for inducible high expression
- A double terminator for antisense inhibition avoidance
- Twelve high-expression genetic circuits assembled using the MoClo Golden Gate system
For more information about each of these components, explore the registry pages below!
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765