Difference between revisions of "Part:BBa K3038002:Experience"
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https://static.igem.org/mediawiki/parts/b/bb/T--Poitiers--TesA_amplification-tab3.png | https://static.igem.org/mediawiki/parts/b/bb/T--Poitiers--TesA_amplification-tab3.png | ||
| + | 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 | ||
Expression | Expression | ||
Revision as of 10:09, 16 October 2019
Applications of BBa_K3038002
Characterization of TesA :
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
Expression
Activity
User Reviews
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