Team:Bio Without Borders/Design

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• JUDGING FORM

Creating Factor C

We used an NEBuilder kit to combine two sequences of the Limulus polyphemus that IDT sent us, as the whole sequence was too large to send as one part. We first confirmed that we could at the j04450 insert into the pSB1C3 backbone for submission. Once we confirmed this process, we then used the NEBuilder kit to put together the two G-Blocks of the Factor C sequence inside the pSCB1C3 backbone.

Creating a linker protein of the cellulose-binding domain (CBD) and GFP

We decided to revive two previous iGem parts, the cellulose binding domain (CBD- BBa_K1478001) and green fluorescent protein (GFP- BBa_E0440) in order to create the fusion protein, which would also include our double Isoleucine linker bond, which Factor C will ideally cleave in addition to itself. This would also function as the reporter protein which would demonstrate Factor C's ability to cleave. In our design, the CBD would be bound to a paper substrate, so that when cleaved in a test solution, the GFP would migrate via capillary action up the paper. In addition to a visual read-out, we would also quantify and with a fluorimeter for the assay. In the middle of our research, we realized that there existed a CBD-GFP fusion protein, and so we then used that part and ligated the fusion linker within that.

Expressing Factor C through Pichia pastoris

After making our parts, we wanted to express the parts in an easy model organism like pichia. IDT came back to us with a complete sequence, and so we no longer needed to use the NEBuilder. We decided to move forward in our experiment and use the electroporator to insert the Factor C within the yeast.