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| | ==Description== | | ==Description== |
| − | Alcohol dehydrogenase, ADH or ADR N-term, is a BioBrick C-Myc and 6-His tagged in N-term.<br/> | + | Alcohol dehydrogenase, ADH or ADR C-term, is a BioBrick C-Myc and 6-His tagged in C-term.<br/> |
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| | ADR is a thermophilic NAD+ dependent alcohol dehydrogenase. | | ADR is a thermophilic NAD+ dependent alcohol dehydrogenase. |
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| | Secondary alcohol + NAD+ = ketone + H+ + NADH | | Secondary alcohol + NAD+ = ketone + H+ + NADH |
| | EC:1.1.1.1 | | EC:1.1.1.1 |
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| − | ===Alcohol dehydrogenase; Short= ADH or ADR===
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| − | ===Thermophilic NAD+-dependent alcohol dehydrogenase===
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| − | Bears mainly an ethanol-dehydrogenase activity.
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| − | ===Reaction===
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| − | a secondary alcohol + NAD+ = a ketone + H+ + NADH
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| − | EC:1.1.1.1
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| − | ===Protein Sequence ===
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| − | MVKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVK
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| − | SIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGV
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| − | TTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQ
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| − | VGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGK
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| − | VRPIVETAELEEINEVFERMEKGKINGRIVLKLKEDEQKLISEEDLNSAVDHHHHHH
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| − | ===Usage and Biology===
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| − | ===PCR amplification of the PCR products===
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| − | https://2019.igem.org/wiki/images/4/40/T--Poitiers--PCR_amplification_ADR-tab3.jpg
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| − | Electrophoresis photography following deposits on agarose gel 0.8% of enzymatic digestion products. The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder. Lane 1 corresponds to the marker, lane 2 to the digested N-ter,ADR lane 3 to the digested C-ter ADR and lane 4 to the digested plasmid pSB1A3.
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| − | ===Plasmid construction===
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| − | https://2019.igem.org/wiki/images/5/5b/T--Poitiers--plasmid_construction_ADR-tab3.jpg
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| − | Design of ADR N-ter/pSB1A3 and ADR C-ter/pSB1A3 with Geneious software. This map shows the pBAD promoter and its terminator flanking the coding sequence of the ADR protein. Also present in N-ter or C-ter are 6-His and c-myc tag. Finally, in the plasmid is present and ampicillin resistance cassette.
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| − | ===Expression of the CMYC-6HIS-ADR and ADR-CMYC-6HIS recombinant proteins===
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| − | https://2019.igem.org/wiki/images/6/67/T--Poitiers--recombinantexpression_ADR-tab3.png
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| − | NI : Not induced
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| − | I: Induced
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| − | After sequencing, induction was performed on the thermocompetent bacteria JM109. The objective was to verify if the cloned gene leads to the production of a protein. The expected size of the ADR protein is 40 kDa. A very strong expression of the ADR protein was observed at this size when the pBAD promoter is induced with arabinose. The gene has therefore been correctly cloned into the strain and the protein is produced.
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| − | ===Activity===
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K3038001 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K3038001 parameters</partinfo>
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Revision as of 12:45, 18 October 2019
Alcohol dehydrogenase - Geobacillus stearothermophilus_Cmyc and 6His tags_Cterminal
Description
Alcohol dehydrogenase, ADH or ADR C-term, is a BioBrick C-Myc and 6-His tagged in C-term.
ADR is a thermophilic NAD+ dependent alcohol dehydrogenase.
This enzyme bears mainly an ethanol-dehydrogenase activity.
GenBank
ADR : GenBank: P42327
https://www.ncbi.nlm.nih.gov/protein/P42327
Protein Sequence
MEQKLISEEDLNSAVDHHHHHHVKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGD
WPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDG
GYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMG
LNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLV
VVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKI
NGRIVLKLKED
Reaction
Primary alcohol + NAD+ = Aldehyde + NADH + H+
Secondary alcohol + NAD+ = ketone + H+ + NADH
EC:1.1.1.1
