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
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Unknown
• 1000
  COMPATIBLE WITH RFC[1000]

Design

Fig.x:RecA intein behavior when an EDC is present.

This RecA intein ESR1 biosensor consists of two inteins capable of joining and separating from two protein fragments. Considering this, the ESR1 biosensor gene coding for the hERalpha protein was introduced with its respective linker, in such a way that when an endocrine disruptor binds to the biosensor, the inteins join and separate with the rest of the protein, removing the linker, the biosensor and the two inteins. The two endogenous fragments encode the chromoprotein AmilCP [BBa_K592009].

RecA intein was build from the first 110 and last 58 aminoacids from the wildtype RecA full-lenght intein. Moreover two aminoacid mutationes (Val67Leu and Asp111Glu) to make the RecA intein a stable protein.

References

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.

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.