Part:BBa_K4260111

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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. </html>

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.

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.

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.