Coding
CMYC 6HIS

Part:BBa_K3038000

Designed by: Marine LAVAUD   Group: iGEM19_Poitiers   (2019-10-08)
Revision as of 11:44, 18 October 2019 by Mlavaud (Talk | contribs) (→‎Alcohol dehydrogenase; Short = ADH or ADR)

Description

GenBank

ADR : GenBank: P42327
https://www.ncbi.nlm.nih.gov/protein/P42327

Reaction

Primary alcohol + NAD+ = Aldehyde + NADH + H+ Secondary alcohol + NAD+ = ketone + H+ + NADH EC:1.1.1.1

Protein Sequence

MEQKLISEEDLNSAVDHHHHHHVKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGD WPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDG GYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMG LNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLV VVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKI NGRIVLKLKED


Usage and Biology

The alcohol deshydrogenase catalyze the oxidation reaction of many alcohols. In our case, it allows to oxidized fatty acids. ADH bacteria have a reverse function to that describe in the human body. It then produces alcohol bu generating NAD+. This is called alcoholic fermentation.

Applications of BBa_K3038000

PCR amplification of the PCR products

T--Poitiers--PCR_amplification_ADR-tab3.jpg

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

T--Poitiers--plasmid_construction_ADR-tab3.jpg

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

T--Poitiers--recombinantexpression_ADR-tab3.png

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None