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Team:Tuebingen/Project/Inverter
From 2013.igem.org
Once progestin binds to the mPR the FET3 gene promoter (Pfet3) will be repressed by an endogenous signaling molecule from the PAQR-pathway of our chassis (S. cerevisiae). As a result the expression of downstream genes of Pfet3 will stop. In order to receive a positive feedback when progestin is detected we need to invert the receptor signal – therefore we have combined Pfet3 with a repressor that represses the promoter of our reporter.
The repressor that represses the promoter of our reporter – i.e. the inverter of the receptor signal – is rather unspectacular and was mainly employed for its simplicity. Hoping that there will not be any interference by normal cellular metabolism we decided to use endogenous repressors of S. cerevisiae and came up with the following two inverter-systems:
In Saccharomyces cerevisiae the expression of SUC2 genes (which encode invertase) is heavily repressed by high levels of glucose (Lutfiyya and Johnston, 1996). The proteins which repress SUC2 genes in this case are MIG1 and MIG2 whereby MIG1 is a stronger repressor than MIG2 (Lutfiyya and Johnston, 1996). MIG1 and MIG2 both are zinc-finger-containing repressor proteins with binding-sites in the SUC2 promoter. MIG1 has its highest affinity to the GC-rich SUC2-A site which is between position -505 and -483 in the SUC2 promoter (Nehlin and Ronne, 1990, Lutfiyya and Johnston, 1996). The release of repression is mediated by the protein kinase SNF1 (sucrose nonfermenting) in yeast (Celenza and Carlson, 1986, Carlson, 1998). According to Nehlin and Ronne (1990) and Chu et al. (1986) MIG1 is located on yeast chromosome 7.
For the first inverter-system we basically use the MIG1 gene and the SUC2 promoter (Psuc2) whereby the MIG1 gene is downstream of Pfet3 and Psuc2 is upstream of the reporter. Thus, as long as MIG1 is expressed the reporter is repressed via Psuc2 repression.
There are many genes in yeast that are regulated by oxygen supply as a means to adapt to fluctuations in oxygen tension (Lowry and Zitomer, 1988). One of these genes is the ANB1 gene: under aerobic conditions the ANB1 gene is regulated by heme while it is expressed under anaerobic conditions (Lowry and Zitomer, 1984). Under aerobic conditions “the ROX1 gene product mediates the heme regulation of the ANB1 gene” (Lowry and Zitomer, 1988). Lowry and Zitomer (1988) found that this repression is regulated by the synthesis and elimination of ROX1 transcripts depending on the presence of oxygen whereby aerobic conditions induce the synthesis of heme which in turn stimulates the expression of ROX1. “ROX1 is a member of the HMG family of DNA-binding proteins” (Balasubramanian et al., 1993) and binds to a “12-bp consensus sequence repeated twice in two of the ROX1-responsive operators of ANB1” (Lowry et al., 1990). According to Lowry et al. (1990) the operator sequences (which are the targets for ROX1) are located in the upstream region of the ANB1 gene. ROX1 is located on yeast chromosome 16 (Balasubramanian et al., 1993).
For the second inverter-system we employ the ROX1 gene and the ANB1 upstream region/promoter (Panb1) whereby the ROX1 gene is downstream of Pfet3 and Panb2 is upstream of the reporter. Thus, as long as ROX1 is expressed the reporter is repressed via Panb1 repression.
The advantage of these systems is their robustness: there might be other (maybe even unknown) factors that play a role in each repressor gene’s functionality like the heme which mediates ROX1 repression or SNF1 which is important for the release of repression of MIG1 but since both repressor-systems are native to yeast all additionally required factors are available.
References
BALASUBRAMANIAN, B., LOWRY, C. V. & ZITOMER, R. S. 1993. The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif. Mol Cell Biol, 13, 6071-8.
CARLSON, M. 1998. Regulation of glucose utilization in yeast. Current Opinion in Genetics & Development, 8, 560-564.
CELENZA, J. & CARLSON, M. 1986. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science, 233, 1175-1180.
CHU, G., VOLLRATH, D. & DAVIS, R. 1986. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science, 234, 1582-1585.
LOWRY, C. V., CERDAN, M. E. & ZITOMER, R. S. 1990. A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae. Mol Cell Biol, 10, 5921-6.
LOWRY, C. V. & ZITOMER, R. S. 1984. Oxygen regulation of anaerobic and aerobic genes mediated by a common factor in yeast. Proceedings of the National Academy of Sciences, 81, 6129-6133.
LOWRY, C. V. & ZITOMER, R. S. 1988. ROX1 encodes a heme-induced repression factor regulating ANB1 and CYC7 of Saccharomyces cerevisiae. Mol Cell Biol, 8, 4651-8.
LUTFIYYA, L. L. & JOHNSTON, M. 1996. Two zinc-finger-containing repressors are responsible for glucose repression of SUC2 expression. Mol Cell Biol, 16, 4790-7.
NEHLIN, J. O. & RONNE, H. 1990. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. Embo j, 9, 2891-8.
