Jay Yung

Jay Yung

Aug 29, 2019

Group 6 Copy 172
0

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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About This Project

Chemically induced dimerization (CID) is the process of protein dimerization in the presence of a small molecule. CID allows for highly specific molecule sensing and flexible custom protein design, offering a versatile platform for sensing molecules. However, CID is underutilized in biosensing. We hypothesize that a CID binder-based detection assay can be developed and used to accurately quantify a variety of important small molecules with applications in research, medicine, and more.


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