Difference between revisions of "Part:BBa K2539400"

Revision as of 05:26, 11 October 2018

PalcA-Regulated ALDH2*1 Expression Construct

This construct uses the inducible promoter PalcA (BBa_K2092002). PalcA is activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001). PalcA is activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001), and was originally isolated from the fungus Aspergillus nidulans (Panozzo et al., 1997). 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

The PalcA promoter is placed in front of a strong RBS (BBa_B0034), the human ALDH2*1 sequence (BBa_K2539150), and double terminator (BBa_B0015). This places the expression of ALDH2*1 under the control of the PalcA promoter.

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_K2539400 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2.9 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.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 274
Illegal BglII site found at 2116
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1174
Illegal NgoMIV site found at 1317
Illegal NgoMIV site found at 1830
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1236
Illegal SapI.rc site found at 1733

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2539400 short</partinfo>
-A regulated ALDH2*1 expressing construct that employs the ethanol and alcR induced promoter (BBa_K2092002) and a strong RBS (BBa_B0034) to regulate expression of ALDH2*1, a mitochondrial enzyme responsible for the oxidation of acetaldehyde into acetate. The sequence is then terminated with a double terminator (BBa_B0015) to ensure the end of transcription.
+This construct uses the inducible promoter PalcA (BBa_K2092002). PalcA is activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001).  PalcA is activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001), and was originally isolated from the fungus Aspergillus nidulans (Panozzo <i>et al.</i>, 1997).
+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 <i>et al.</i>, 2005; Farrés <i>et al.</i>, 1994).
+<b><font size="+1">Construct Design</font></b>
+The PalcA promoter is placed in front of a strong RBS (BBa_B0034), the human ALDH2*1 sequence (BBa_K2539150), and double terminator (BBa_B0015). This places the expression of ALDH2*1 under the control of the PalcA promoter.
+https://static.igem.org/mediawiki/parts/4/42/T--TAS_Taipei--400construct.jpg
+<b><font size="+1">PCR Check Results</font></b>
+The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
+https://static.igem.org/mediawiki/parts/a/a2/T--TAS_Taipei--300pcr.jpg
+<b>PCR check for BBa_K2539400 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2.9 kb.</b>
+<b><font size="+1">References</font></b>
+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.
+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.