Coding
CMYC 6HIS

Part:BBa_K3038000

Designed by: Marine LAVAUD   Group: iGEM19_Poitiers   (2019-10-08)
Revision as of 12:34, 18 October 2019 by Mlavaud (Talk | contribs) (→‎Manipulations)

Description

Alcohol dehydrogenase, ADH or ADR N-term, is a BioBrick C-Myc and 6-His tagged in N-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

Usage and Biology

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

Design

Thanks to Geneious software we have designed a gene with a promoter, a C-Myc and 6-His tag and a terminator. The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. The allows to purify and detect the protein in the host strain by using Ni-NTA columns or specific antibodies.

Manipulations

PCR amplification

Following the design of the synthetic gene, it is amplified by PCR thanks to the design of primers upstream and downstream of the sequence.

T--Poitiers--PCR_amplification_ADR-tab3.jpg

Electrophoresis photography following loads 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 control without DNA, lane 3 to the amplified N-term ADR and lane 4 to the amplified C-term ADR.

Cloning design in pSB1A3

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.

Cloning into pSB1A3

After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1A3 according to the protocol described above. The insert (N-term ADR or C-term) is then ligated into the plasmid.

T--Poitiers--electrophor%C3%A8se_gel4_tab4.png

Electrophoresis photography following loads 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 (left in the figure). Lane 1 corresponds to the marker, lane 2 to the digested N-term ADR, lane 3 to the digested C-term ADR and lane 4 to the digested pSB1A3 plasmid.

Cloning into Thermocompetent cells JM109

The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained.

T--Poitiers--culture_gauche2_tab4.png Clones on a selective LB medium (+ ampicillin 100 µg/mL) following the transformation of thermocompetent cells with the pSB1A3-ADR ligations.
A: Clones obtained from pSB1A3 N-ter ADR ligations.
B: Clones obtained from the pSB1A3 C-ter ADR ligations.

Expression of the CMYC-6HIS-ADR and ADR-CMYC-6HIS recombinant proteins

T--Poitiers--recombinantexpression_ADR-tab3.png

NI : Not induced
I: Induced
M: Marker

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]


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Categories
Parameters
None