Our Progress So Far
As of this post, here are the things the Washington iGEM wetlab has accomplished so far!
Mutagenesis
Informed by simulations, we expect to see the binding properties of the anchor binder to CBD
This is tested through an OCTET bio-layer interferometry assay
So far, we have run 4 different OCTET assays, and we plan to run a few more with high binder concentrations to get a more clear picture of the binding properties.
Biosensor
We have designed coexpression plasmids for the construction of our biosensor system using CID binders.
This plasmid contains the CBD Anchor Binder, linked to a subunit of the Split Luciferase complex, and a CBD Dimer Binder, linked to the other subunit of the Split Luciferase complex.
So far we have assembled this in two ways; as a biobrick through gene synthesis and in the pADL-23c vector through Gibson Assembly
We are currently confirming our plasmids and transforming them into protein expressing strains of E. coli
What's next?
We plan finish up another OCTET soon and confer with our simulations team; based on the results we may try another mutation if we have the time.
For the biosensor, we will perform verification tests to see if our system works at all in the cells. If this is successful, we will move onto to attempting to characterize the sensor through luminescence as a measurement of small molecule concentration.
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