Difference between revisions of "Part:BBa K3038002"

GenBank

TesA : GenBank: EG11542
https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3

Description

Tes A is a Acyl ACP thioesterase gene. Improvement of BB K1472601 : Addition of a 6his Tag at the C-terminal part of the coding sequence.

Reaction

The Acyl ACP thioesterase convert fatty acids ACP or fatty acids CoA into free fatty acids.

Usage and Biology

In order to produce the molecule of interest 2-nonanone, we worked with the Lawrence Berkeley National Laboratory, USA which is working on biofuels and modified E. coli strain and obtain a production of 2-nonanone. This production is possible using free fatty acids as substrate.

Here we present the cloning of thioesterase I (TesA), an enzyme involved in the synthesis of free fatty acids in E. coli .

Design

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 allows to purify and detect the protein in the host strain by using Ni-NTA columns.

https://parts.igem.org/File:T--Poitiers--TesA_design-tab3.jpg

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. 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 deposited 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 specific pairing of the primers.

Electrophoresis gel photography following deposit of TesA PCR products. 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

Cloning design in pSB1A3

The products of digestion are also loaded on the 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.

Cloning into 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 (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

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.

PCR colony screening

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 deposited 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 aspecific band obtained after PCR on the synthetic gene.

Electrophoresis photography following deposits 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 E. coli 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 20 kDa.

Expression

Activity

Sequence and Features

References

Engineering of Bacterial Methyl Ketone Synthesis for Biofuels. Ee-Been Goh,a,c Edward E. K. Baidoo,a,c Jay D. Keasling,a,c,d and Harry R. Beller. Appl Environ Microbiol. 2012 Jan; 78(1): 70–80. doi: 10.1128/AEM.06785-11. PMCID: PMC3255637. PMID: 22038610