Team:Poitiers/Improve
Improve (TesA)
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Here we present the cloning of thioesterase I (TesA), an enzyme involved in the synthesis of free fatty acids in E. coli.
Thanks to Geneious software we have designed a gene with a promoter, and a tag. This part doesn’t have a terminator because its produced to create a composite part with other gene involved in 2-nonanone synthesis. The promoter will therefore be associated with the design of the last gene of the composite part. 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.
This part is already exciting with number. But we decided to improve it by adding a 6-his tag. This allow to purify and detect the protein in the host strain by using Ni-NTA columns.
Photography of designed gene realized with the Geneious software.
Design of TesA C-ter flanked with a tag 6-His and restriction sites EcorI, XbaI, SpeI and PstI + pBAD promoter. This is a 5'-3'-end sequence.
Following the design of the synthetic gene, It is amplified by PCR thanks to the design of primers upstream and downstream of the sequence. 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 (TesA) is then ligated into the plasmid.
The PCR product as well as the digestion products are loaded on 0.8% agarose gel. In well 2, the TesA tagged with 6 his in C-ter amplified by PCR. The most intense band observed corresponds to the size expected for TesA around 900 pb. Another band, this time very weak, is visible below 400 pb. This band may be due to a non specific pairing of the primers.
Photography of designed gene realized with the Geneious software.
The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is NEB 1 kb Plus DNA Ladder.
The products of digestion are also loaded on gel. In well 2 we see the purified PCR TesA product. There is little DNA loss here, which is encouraging. Wells 3 and 4 respectively show the digestion of the plasmid and the TesA gene by the restriction enzymes EcoRI and PstI. This is to form cohesive ends between the two. We obtain bands at the expected sizes, about 2200 pb for the plasmid and 900 pb for the synthetic gene TesA.
It is important to note, however that agarose gel migration does not verify the effectiveness of digestion. Indeed, since the restriction sites are at the end of the sequences, only a few base pairs have been removed on either side. The resolution of an agarose gel does not make it possible to observe the size of the fragments so precisely. This step makes it possible to ensure that we did not have a loss of DNA during experiments.
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 purified PCR product, lane 3 to the digested pSB1A3 plasmid and lane 4 to the digested TesA synthetic gene.
Finally, the ligation is performed. To model the construction, we used the Geneious software which allowed us to assemble the sequence of the plasmid pSB1A3 as well as that of ADR N-ter or ADR C-ter inserts at the cloning site. We can then know the size of the construction and visualize the restriction site present on the latter.
Modelization of the ligation between TesA/pSB1A3 with Geneious software.
This map shows the pBAD promoter upstream the coding sequence of the ADR protein. Also present in C-ter the 6-His tag. Finally, in the plasmid is present an ampicillin resistance cassette.
The thermocompetent E. coli JM109 bacteria are then transformed and clones are obtained. Different volumes of transformed bacteria are spread on Petri dish with selective medium. The number of clones obtained is consistent with the proportion of bacteria spread on the petri dishes.
Clones on a selective LB medium (+ ampicillin 100 µg/mL) following the transformation of thermocompetent cells with the pSB1A3-TesA ligation and a control plasmid.
A&B: Clones obtained from pSB1A3-TesA. C: Clones obtained from a control plasmid pSB1C3-Red (iGEM parts).
After bacterial transformation, colony PCR is performed with the forward primer of the TesA gene and a reverse primer of the plasmid. 24 clones are tested. The PCR products are loaded on 0.8% agarose gel.
Clones 1, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 17, 18, 19, 21, 23 and 24 have the right profile, an insert-vector fragment of 1100 pb. Wells 2 and 11 show nothing so they probably did not integrate the ligation products.
Wells 10, 16, 20 and 22 seem to have incorporated the non specific band obtained after PCR on the synthetic gene.
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. Lane 1 to 10 corresponds to colony PCR performed on TesA/pSB1A3 ligation, lane 11 to 24 corresponds to colony PCR performed on TesA/pSB1A3.
Clones with the right profile are returned to liquid culture and minipreparations are performed. In order to avoid any risk of point mutation, sequencing is performed with the plasmid primer.
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 TesA protein is about 20 kDa. An expression of the TesA 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 12% photography following the induction of JM109 with arabinose after 4 hours of culture.
Coloring with coomassie blue. The lane 1 to the marker. The lane 2 to 5 correspond to induce (I) or non induce (NI) cultures transformed with TesA/pSB1A3.
The last step consists in evaluating the enzymatic activity of the TesA protein in vitro.