Part:BBa_K4260111:Design
Design
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
RecA intein ESR1 biosensor consists of two inteins capable joining two protein fragments and separating from them. The ESR1 biosensor gene coding for the hERalpha protein was introduced with its respective linker, with the purpose of separating the N-terminal and C-terminal RecA inteins in the presence of EDCs; when the ESR1 biosensor binds to an EDC, the inteins may carry protein spicing, ligate the two N and C exteins and separate the ESR1 biosensor, ESR1 linker and the two N and C extein complex. The two endogenous fragments encode the chromoprotein AmilCP [BBa_K592009].
RecA intein was built from the first 111 and last 58 amino acids from the wildtype RecA full-length intein [1]. Moreover, a mutation was carried (Val67Leu) to make the RecA mini intein a stable protein since the central endonuclease domain was missing [2] [3]. The functionality of the Barcelona 2020 team's inteins from the biobrick [BBa_K348400] was verified to check the functibility of the HERα enzyme to be used in the biosensor, since it is employed as a receptor enzyme for EDCs.
A solubility tag was added for easier usage of the coding protein: the OmpA solubility tag (Outer membrane Protein A) allows to redirect a recombinant protein to the outer membrane. With this in consideration, the presence of the protein in the outer membrane may allow for an increment of interactions between the ESR1 biosensor and the EDC. This solubility tag was retrieved from literature because of its efficiency as periplasmic expression signal peptide [4].
Linkers are short amino acid sequences that act as spacers between protein domains within a protein. The ones containing Glycines are flexible, separating domains and mostly, creating covalent bonds between proteins. Adding Serine as a polar residue reduces linker protein interaction preserving protein function of both ESR1 biosensor and the RecA N-intein and C-intein [5][6].
The whole coding sequence consists of the OmpA solubility tag for better interaction between the biosensor and EDCs, the first 100 amino acids from the AmilCP blue chromoprotein [BBa_K592009], the N-terminal RecA mini intein consisting of the first 111 amino acids from Mycobacterium Tuberculosis (Mtu) RecA intein with optimized codons for E. Coli with a mutation (V67L) for stabilizing the structure that had been perturbed without a central endonuclease domain [6], the ESR1 linker to avoid interactions between proteins, the ESR1 biosensor, the C-terminal RecA mini intein consisting of the last 58 amino acids of Mtu RecA intein, and the rest of the AmilCP blue chromoprotein.
References
[1] Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., & Belfort, M. (1999). A genetic system yields self-cleaving inteins for bioseparations. Nature biotechnology, 17(9), 889-892.
[2] Gierach, I., Li, J., Wu, W. Y., Grover, G. J., & Wood, D. W. (2012). Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and β-1. FEBS open bio, 2, 247-253.
[3] Van Roey, P., Pereira, B., Li, Z., Hiraga, K., Belfort, M., & Derbyshire, V. (2007). Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue. Journal of molecular biology, 367(1), 162-173.
[4] TecCEM 2021 https://2021.igem.org/Team:TecCEM
[5] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman. (2014) Design and characterization of structured protein linkers with differing flexibilities. Protein Engineering, Design and Selection, Volume 27, Issue 10, Pages 325–330. https://doi.org/10.1093/protein/gzu043
[6] Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. doi:10.1016/j.addr.2012.09.039