Part:BBa_K2539450
AlcR and PalcA-Regulated ALDH2 Expression Construct
This construct combines two of our submitted composite parts: a constitutively expressed alcR construct (BBa_K2539300) and a PalcA-regulated ALDH2*1 expression construct (BBa_K2539400). PalcA is an inducible promoter (BBa_K2092002), which can be activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001). It was originally isolated from the fungus Aspergillus nidulans (Panozzo et al., 1997). AlcR is a gene found in Aspergillus nidulans which encodes for a regulatory protein (Panozzo et al., 1997). AlcR can bind to the alcA and aldA promoters (PalcA and PaldA, respectively). In the presence of both AlcR and ethanol or threonine, genes downstream of these promoters are expressed (Felenbok et al., 1988). ALDH2*1 is the wild type form of human mitochondrial aldehyde dehydrogenase (ALDH2), which functions in alcohol metabolism to convert the toxic intermediate, acetaldehyde, into acetate (Larson et al., 2005; Farrés et al., 1994).
Construct Design
This construct constitutively express alcR, a regulatory protein necessary for the activation of PalcA. We use a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the alcR sequence (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription. Behind this, the PalcA promoter is placed in front of a strong RBS (BBa_B0034), the human ALDH2*1 sequence (BBa_K2539150), and a double terminator (BBa_B0015). Together, this construct expresses alcR, which can activate the PalcA promoter and the expression of ALDH2*1 if ethanol is present.
PCR Check Results
The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
PCR check for BBa_K2539450 (E) using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 5.7 kb.
References
Farrés J, Wang X, Takahashi K, Cunningham SJ, Wang TT, Weiner H. (1994). Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase. J Biol Chem. 13;269(19):13854-60.
Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW. (1988). The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.
Larson HN, Weiner H, Hurley TD. (2005). Disruption of the Coenzyme Binding Site and Dimer Interface Revealed in the Crystal Structure of Mitochondrial Aldehyde Dehydrogenase “Asian” Variant. J Biol Chem. 280(34):30550-6.
Panozzo C, Capuano V, Fillinger S, 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. J Biol Chem. 5;272(36):22859-65.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2611
Illegal BglII site found at 3075
Illegal BglII site found at 4917
Illegal BamHI site found at 1615
Illegal BamHI site found at 2353 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3975
Illegal NgoMIV site found at 4118
Illegal NgoMIV site found at 4631
Illegal AgeI site found at 1319
Illegal AgeI site found at 1447
Illegal AgeI site found at 1978 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4037
Illegal SapI.rc site found at 4534
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