Difference between revisions of "Part:BBa K3038000"

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.
Although E. coli has a native ADH, we decided to clone this one for better results. Indeed, the alcohol dehydrogenase of Geobacillus stearothermophillus has a better affinity for the substrate but also a better enzymatic activity. The use of the latter could therefore optimize our yields of production.

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 tag 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.

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 DNA 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.

The expected size of the fragment is about 1600 pb. The band obtained correspond to this size.

Enzymatic digestion

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.

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.

Ligation in pSB1A3

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 E. coli Thermocompetent cells JM109

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

Clones on a selective LB medium (+ ampicillin 100 µg/mL) following the transformation of E. colithermocompetent 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.

PCR colony screening

After bacterial transformation, colony PCR is performed with the forward primer of the ADR gene and a reverse primer of the plasmid. 12 clones of each condition (ADR N-ter/pSB1A3 and ADR C-ter/pSB1A3) are tested. The PCR products are loaded on 0.8% agarose gel.

Electrophoresis photography following loads on agarose gel 0.8% of colony PCR 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 to 12 corresponds to colony PCR performed on ADR N-ter/pSB1A3 ligation, lane 13 to 24 corresponds to colony PCR performed on ADR C-ter/pSB1A3.

Clones 4, 5, 10, 11 and 12 have the right profile, an insert-vector fragment of 1800 pb. Wells 13 to 24 show PCR products on clones transformed with ADR C-ter/pSB1A3. Clones 13, 21 and 22 have the right profile, an insert-vector fragment of 1800 pb.

Control enzymatic digestion

Clones with the right profile are returned to liquid culture and minipreparations are performed. Enzymatic digestion is carried out with BamHI and PstI restriction enzyme. The expected band sizes are 2300 and 1400 pb.

Electrophoresis photography following loads on agarose gel 0.8% of enzymatic digestion products by BamHI and PstI.
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 to 5 corresponds to enzymatic digestion product of ADR N-ter/pSB1A3, lane 7 to 9 corresponds to enzymatic digestion product of ADR C-ter/pSB1A3.

Wells 1 to 5 comprise clones 4, 5, 10, 11 and 12 transformed with ADR N-ter/pSB1A3. Wells 7 to 9 contain clones 13, 21 and 23 transformed with ADR C-ter/pSB1A3. All present the right profile of digestion. This experiment therefore confirms the plasmid constructs. In order to avoid any risk of point mutation, sequencing is performed with the plasmid primer.

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

After sequencing, induction is performed on the thermocompetent bacteria JM109. The objective is 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 is 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.

SDS Page 8% photography following the induction of JM109 with arabinose after 4 hours of culture.
Coloring with coomassie blue. The lane 1 to 4 correspond to induce or non induce cultures transformed with ADR N-ter/pSB1A3. Lane 6 to 8 correspond to induce or non induce cultures transformed with ADR C-ter/pSB1A3. NI : Not induced
I: Induced

M: Marker

The last step consist in evaluating the enzymatic activity of the protein in vitro.

Activity

References

Holland-Staley, Carol A.; Lee, KangSeok; Clark, David P.; Cunningham, Philip R. (2000) Aerobic Activity of Escherichia coli Alcohol Dehydrogenase Is Determined by a Single Amino Acid. In : Journal of Bacteriology, vol. 182, n° 21, p. 6049–6054. PMCID: PMC94738
Shim; Eun, Jung; Sang, Hoon, Jeon; Kwang, Hoon, Kong (2003) Overexpression, Purification, and Biochemical Characterization of the Thermostable NAD-dependent Alcohol Dehydrogenase from Bacillus stearothermophilus. In : J. Microbiol. Biotechnol., 13(5), p. 738–744.

Sequence and Features