Part:BBa_K5477006
pPDC1 - Lex6Op - pENO1 bidirectional synthetic promoter with Lex6Op
The pPDC1 core promoter drives the expression of the PDC1 gene, one of the three pyruvate decarboxylase isozymes in Saccharomyces cerevisiae. PDC1 plays a role in fermentation, catalyzing the conversion of pyruvate to acetaldehyde. pPDC1 is also involved in amino acid catabolism and is regulated by glucose, ethanol, and autoregulatory mechanisms (1) (3) (5).
The pENO1 core promoter drives the expression of ENO1, which encodes Enolase I, an enzyme in glycolysis that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (2) (4).
In the Lex6Op bidirectional system, pPDC1 serves as a promoter, working in tandem with pENO1 to drive the expression of two different genes in opposite orientations (6). The Lex6Op operator cassette, composed of six tandem LexA operator sites, regulates the bidirectional activation of both promoters (6). This bidirectional promoter is activated by a receptor module with LexA DNA-binding domain. From a study by Zhou et al. 2022, when a LexA is fused with LexA-ERα BBa_K5477013 or LexA-mERα BBa_K5477014, this acts as a chimeric activator and binds to the Lex6Op sequence, it activates transcription through both pENO1 and pPDC1.
Based on the aforementioned study, a receptor module BBa_K5477029 and reporter module BBa_K5477031 were combined. Another composite part, a detoxification module was integrating this synthetic promoter BBa_K5477001. These three modules comprise the SUPERMOM device BBa_K5477046. Upon presence of BPA, the contaminant will bind to the chimeric activator LexA-mERα BBa_K5477014 . The chimeric activator will bind to the Lex6Op in the reporter module triggering the transcription of NanoLuc and also the detoxification module, since it contains the Lex6Op between the two promoters of UGT2B15 and UDPD.
| Constructs | Module | Composite Part Name |
|---|---|---|
| pRET2-LexA-mERα (LBD) | Receptor module | BBa_K5477029 |
| Lex6Op-pLEU2-NanoLuc | Reporter Module | BBa_K5477031 |
| UDPD-pPDC1-Lex6Op-pENO1-UGT2B15 | Detoxification Module | BBa_K5477040 |
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]
References
1. Boer VM, de Winde JH, Pronk JT, Piper MD. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J Biol Chem. 2003 Jan 31;278(5):3265-74. doi: 10.1074/jbc.M209759200. Epub 2002 Oct 31. PMID: 12414795.
2. Entian KD, Meurer B, Köhler H, Mann KH, Mecke D. Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae. Biochim Biophys Acta. 1987 Feb 20;923(2):214-21. doi: 10.1016/0304-4165(87)90006-7. PMID: 3545298.
3. Kellermann E, Seeboth PG, Hollenberg CP. Analysis of the primary structure and promoter function of a pyruvate decarboxylase gene (PDC1) from Saccharomyces cerevisiae. Nucleic Acids Res. 1986 Nov 25;14(22):8963-77. doi: 10.1093/nar/14.22.8963. PMID: 3537965; PMCID: PMC311923.
4. Klein CJL, Olsson L, Nielsen J. Glucose control in Saccharomyces cerevisiae: the role of Mig1 in metabolic functions. Microbiology (Reading). 1998 Jan;144 ( Pt 1):13-24. doi: 10.1099/00221287-144-1-13. PMID: 9467897.
5. Pronk JT, Yde Steensma H, Van Dijken JP. Pyruvate metabolism in Saccharomyces cerevisiae. Yeast. 1996 Dec;12(16):1607-33. doi: 10.1002/(sici)1097-0061(199612)12:16<1607::aid-yea70>3.0.co;2-4. PMID: 9123965.
6. Rantasalo A, Czeizler E, Virtanen R, et al. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS One. 2016;11(2):e0148320. Published 2016 Feb 22. doi:10.1371/journal.pone.0148320
7. Zhou, T., Liang, Z. & Marchisio, M.A. Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol. Sci Rep 12, 21791 (2022). https://doi.org/10.1038/s41598-022-26195-x
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