Part:BBa_K3038002:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

https://parts.igem.org/File:T--Poitiers--TesA_amplification-tab3.png Expression

Activity

User Reviews

UNIQaa8bf80e1ca86830-partinfo-00000000-QINU UNIQaa8bf80e1ca86830-partinfo-00000001-QINU