Team:USP-EEL-Brazil/Design
Design
The sequences of our target genes were found in the CAZy data bank, where laccases are identified as enzymes from the Auxiliary Activity Family 1 (AA1):
- Laccase from Pleurotus ostreatus NRRL0366:3471 pb – 533 aa
- Laccase from Phoma sp. UHH 5-1-03 (DSM 2245):3073 pb – 607 aa
To analyze and remove the introns, we used the FGENESH tool from Softberry. After that, to verify if the gene sequence expressed a functional protein we used the Translate toolfrom Expazy. There we confirmed our sequences as correct. Once our laccases are from fungi and we plan to express them into a bacterial chassis, we had to remove the signal peptide. To do so, we used the SignalP 4.1 tool from DTU Bioinformatics .
Completed this process we removed any restriction sites for EcoRI, NotI, PstI and SpeI from the gene. For the synthesis, we inserted an RBS sequence and the biocrick prefix and suffix, obtaining the following gene features:
Phoma sp:
Pleurotus ostreatus:
Our final biobrick construct constituted on the following parts:
| PART NAME | INFORMATION |
|---|---|
|
Promotor Lacl
|
Obtained from the iGEM Registry: BBa_R0010 and found on the 2018 DNA Distribution Kit |
|
RBS
|
Sequence obtained from iGEM Registry BBa_B0030 and synthetised with the gene. |
|
Laccase phoma
|
Designed and synthetised by the team |
|
Laccase pleurotus ostreatus
|
Designed and synthetised by the team |
|
Terminator
|
Found on the pSBIC3 backbone |
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