Team:Manchester/Description/mechanism2
Inducible Gene Switch
How it works?
alcR
alcR is a positive regulatory gene in the ethanol regulon of filamentous fungus Aspergillus nidulans (A.nidulans). It encodes a protein that acts as a transcription factor which would bind to its target promoters alcA (alcAP) and aldA. The expression of the downstream gene of alcAP is strongly induced through the positive transcriptional regulator AlcR protein by various substrates such as ethanol and threonine. For our project, we were interested in the ability of the AlcR protein, under the influence of ethanol, to initiate transcription of chromoproteins by binding to specific sites on the alcAP [1]. The chromoproteins used were from previously characterised BioBricks by 2013 iGEM Uppsala-Sweden: amilCP with RBS ( BBa_K1033930) and spisPink with RBS (BBa_K1033925).
AlcR is also known as a zinc binuclear cluster activator as it contains a DNA-binding domain belonging to the C6 zinc binuclear cluster family. AlcR is unique in that it can bind to symmetric and asymmetric DNA sites with the same apparent affinity and also bind to single site with high affinity [2]. Our project focuses on the ability of AlcR to bind to its binding sites on alcAP. alcR is a new BioBrick we have characterised this year (BBa_K2092001).
alcA promoter (alcA P)
alcA P is one of the strongest inducible promoters in A.nidulans and is widely used to overexpress proteins [2]. Based on our references, we found that the number and the position of the AlcR binding sites on alcAP are crucial for the strength of transcriptional activation of alcAP. Hence, we took the factors into account when deciding which alcAP variants to use for our system. There are evidences that show two sites, either in direct or inverted orientation, are necessary for full transcriptional activation of alcAP ,sup>[3].
Our alcAP variants were inspired by a research done in creating a functional, chemically inducible gene switch for monocotyledonous plant sugar cane [3] and Escherichia coli [4]. alcA 1P is the native variant which is a previously characterised BioBrick by 2011 iGEM DTU-Denmark (BBa_K678001). We have improved this BioBrick by adding several missing restriction sites on the Prefix and Suffix (BBa_K2092002). alcA 2P is a new BioBrick we have characterised this year (BBa_K2092003 ).
alcA 1P and alcA 2P differ in the binding sites for AlcR. alcA 1P has binding sites abc while alcA 2P contains only binding sites bc. Binding site a contains two direct tandem repeats; binding site b, a palindromic target, contains two inverted tandem repeats while binding site c consists of three half sites with both direct and inverted tandem repeats. The binding sites a, b and c have been previously localized in the alcAP by footprinting experiments and it has also been shown that each AlcR target in the alcAP contributes differently to the activation of the downstream protein expression [3].
Reference
- Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997). The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. Journal of Biological Chemistry, 272(36), 22859–22865.
- Felenbok, B., Sequeval, D., Mathieu, M., Sibley, S., Gwynne, D.I. and Davies, R.W. (1988). The ethanol regulon in Aspergillus nidulans: Characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.
- Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.
- Hemmati, H. and Basu, C. (2015). Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein. Biotechnology & Biotechnological Equipment, 29(6), 1043–1052.
