Part:BBa_K4129100

Background for FunsTF01

FunsTF01 is a synthetic transcription factor (sTF) based on sensor of benzoic acid derivatives (sBAD), which is a sTF in S. cerevisiae (Castaño-Cerezo et. al (2020)). FunsTF01 should initiate the transcription through the 6xLexO minimal promoter. This sTF is the sensing part of the biosensor.

FunsTF01 is a fusion protein consisting of the DNA-binding domain: lexA, ligand sensing domain: HbaR, transactivation domain; B112 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR was a longer version (Ottoz et. al (2014 ) compared to sBAD (Castaño-Cerezo et. al (2020)).

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, lexO (Erill. et al (2003)), and it is the DNA binding domain that interacts with LexO that is used in FunsTF01. HbaR is a transcriptional factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid or in the presence of benzoic acid derivatives (Egland. Et al (2000), Castaño-Cerezo et. al (2020)).The transactivation domain B112 is from E. coli, which were experimentally proven to initiate transcription of a synthetic promoter in S. cerevisiae (Ottoz et. al (2014)). The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport og the protein to the nucleus (Garcia-Bustos et. al (1991)).

References:

Garcia-Bustos J, Heitman J, Hall MN. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83-101. doi: 10.1016/0304-4157(91)90013-m. PMID: 2004116.

Ottoz DS, Rudolf F, Stelling J. Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. Nucleic Acids Res. 2014;42(17):e130. doi: 10.1093/nar/gku616. Epub 2014 Jul 17. PMID: 25034689; PMCID: PMC4176152.

Castaño-Cerezo S, Fournié M, Urban P, Faulon JL, Truan G. Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae. Front Bioeng Biotechnol. 2020 Jan 7;7:372. doi: 10.3389/fbioe.2019.00372. PMID: 31970152; PMCID: PMC6959289.

Egland PG, Harwood CS. HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris. J Bacteriol. 2000 Jan;182(1):100-6. doi: 10.1128/JB.182.1.100-106.2000. PMID: 10613868; PMCID: PMC94245.

Radman M. SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. Basic Life Sci. 1975;5A:355-67. doi: 10.1007/978-1-4684-2895-7_48. PMID: 1103845.

Erill I, Escribano M, Campoy S, Barbé J. In silico analysis reveals substantial variability in the gene contents of the gamma proteobacteria LexA-regulon. Bioinformatics. 2003 Nov 22;19(17):2225-36. doi: 10.1093/bioinformatics/btg303. PMID: 14630651.