Team:BIT-China/Contribution
Abstract
For the Contribution, we completed the experimental characterization and searched for more relevant literature of the previous parts (BBa_K2368003 and BBa_K2368004) and added the data of them to the corresponding BioBricks.
| BioBricks | Codes in the lab |
|---|---|
| BBa_K2368003 | T1R2 |
| BBa_K2368004 | T1R3 |
All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.
Contribution
BBa_K2368003 (T1R2)
The protein encoded by T1R2 gene is a G-protein-coupled receptor with seven trans-membrane domains, and is a component of the heterodimeric amino acid taste receptor T1R1+3 and sweet taste receptor T1R2+3. It was first registered in 2017 as a gene expressing sweet taste receptors.
When we used this Biobrick, we did a more thorough literature research on it, supplementing some of the information related to its domain.There are three distinct domains for each monomer protein: Venus Flytrap domain (VFD) in N-terminal, Cysteine-rich domain (CRD) in downstream of VFD, and seven-layer spiral trans-membrane domain (TMD) in trans-membrane region. The seven-layer helical trans-membrane of TMD is a significant feature of the GPCR family of proteins, and this structure is also considered to be a key structure for interaction with intracellular G proteins. The structure of the intracellular segment is very small C-terminal. The extracellular segment of class C GPCR is very large and has glycosylation modification, while the heterodimer form is maintained between the two monomer proteins through disulfide bonds and some non-covalent interactions.
In addition, in order to test whether T1R2 is localized on the membrane, we provided a new scheme: we decided to attach the fluorescent protein gene to T1R2 and observe the localization of T1R2 on the membrane of Saccharomyces cerevisiae with a fluorescence microscope. We successfully connected eGFP to T1R2 receptor by OE-PCR, and the gel picture is as follows:
Fig 1. T1R2+eGFP gel picture
BBa_K2368004 (T1R3)
The protein encoded by the T1R3 gene is a G-protein-coupled receptor with seven trans-membrane domains. The receptor forms a protein dimer with T1R1 or T1R2. It was also first registered in 2017 as a gene that expresses part of the sweet taste receptor. This year we also used it to complete the construction of umami receptor expression plasmid. In experiment, we observed that E. coli transfected with a plasmid expressing the umami receptor could survive normally on the medium supplemented with ampicillin.
Fig 2. Colonies grown after transferring the umami taste receptor expression plasmid
The plasmid construction was verified by colony PCR. See BBa_K3852005 for details.In addition, we have added some information about its domain in the Parts.
There are three distinct domains for each monomer protein: Venus Flytrap domain (VFD) in N-terminal, Cysteine-rich domain (CRD) in downstream of VFD, and seven-layer spiral trans-membrane domain (TMD) in trans-membrane region. The seven-layer helical trans-membrane of TMD is a significant feature of the GPCR family of proteins, and this structure is also considered to be a key structure for interaction with intracellular G proteins. The structure of the intracellular segment is very small C-terminal. The extracellular segment of class C GPCR is very large and has glycosylation modification, while the heterodimer form is maintained between the two monomer proteins through disulfide bonds and some non-covalent interactions.
Reference
[1]Chéron J, Golebiowski J, Antonczak S, et al. The anatomy of mammalian sweet taste receptors[J]. Proteins-structure Function & Bioinformatics, 2017, 85(2):332.