Difference between revisions of "Part:BBa K3038000"
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===ADR : GenBank: P42327=== | ===ADR : GenBank: P42327=== | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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===Applications of BBa_K3038000=== | ===Applications of BBa_K3038000=== | ||
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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. | 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. | ||
Revision as of 13:10, 16 October 2019
Contents
ADR : GenBank: P42327
https://www.ncbi.nlm.nih.gov/protein/P42327
Alcohol dehydrogenase; Short=ADH or ADR
===Thermophilic NAD+-dependent alcohol dehydrogenase===. Bears mainly an ethanol-dehydrogenase activity.
Reaction
a secondary alcohol + NAD+ = a ketone + H+ + NADH EC:1.1.1.1
Protein Sequence
MEQKLISEEDLNSAVDHHHHHHVKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGD WPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDG GYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMG LNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLV VVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKI NGRIVLKLKED
Usage and Biology
Applications of BBa_K3038000
PCR amplification of the PCR products
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.
Plasmid construction
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.
Expression of the CMYC-6HIS-ADR and ADR-CMYC-6HIS recombinant proteins
NI : Not induced I: Induced
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.
Activity
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]