Difference between revisions of "Part:BBa K4260111"

Revision as of 00:43, 6 October 2022

RecA mini intein mediated ESR1 protein with OmpA signal peptide, linker and AmilCP.

This part encodes an intein mediated biosensor, consisting of a divided AmilCP chromoprotein gene, a mutated intein RecA N-terminal and C-terminal domains and a ESR1 Ligand Binding Domain. The RecA intein comes from Mycobacterium Tuberculosis (Mtu) which has been mutated with the purpose of making it more stable.

Sequence and Features

Design

Fig.1:RecA intein mediated biosensor behavior in the presence endocrine disrupting chemicals (EDCs).

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 build from the first 111 and last 58 aminoacids from the wildtype RecA full-lenght intein [1]. Moreover a mutation was carried (Val67Leu) to make the RecA intein a stable protein [2]. 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.

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.

Fig.2:Coding sequence for RecA intein ESR1 biosensor.

Usage, Composition and Biology

Composition

The RecA mini intein ESR1 biosensor was synthesized in two fragments: the first 533 bases as the first fragment and the last 2764 bases as the second fragment by IDT. An Overlapping PCR was carried, where two homologous sequences get overlapped and amplified with the objective of getting a single DNA strand [7]. Immediately the overlapping PCR was done, a normal amplification PCR was carried. The final PCR product was run into an agarose gel through electrophoresis and purified. The vector used to add the RecA intein biosensor was pJET 1.2/blunt and cloned into DH5-alpha [8].

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

[7] Anna Bahle. (2019). Overlap extension PCR. CEPLAS, Heinrich Heine University. Institute for Synthetic Microbiology. Protocols.io. https://dx.doi.org/10.17504/protocols.io.psndnde

[8] pJET 1.2/blunt vector: https://www.snapgene.com/resources/plasmid-files/?set=basic_cloning_vectors&plasmid=pJET1.2

Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in microbiology, 5, 63.

Shah, N. H., & Muir, T. W. (2014). Inteins: nature's gift to protein chemists. Chemical science, 5(2), 446-461.

Shingledecker, K., Jiang, S. Q., & Paulus, H. (1998). Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments. Gene, 207(2), 187-195.

Davis, E. O., Sedgwick, S. G., & Colston, M. J. (1991). Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. Journal of bacteriology, 173(18), 5653-5662.