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):

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 biobrick prefix and suffix, obtaining the following gene features:

Laccase Phoma sp.(Lac PH):

Laccase Pleurotus ostreatus (Lac PL):

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 sp. (Lac PH)

 Designed and synthetised by the team

Laccase Pleurotus ostreatus (Lac PL)

 Designed and synthetised by the team

Terminator

 Found on the pSBIC3 backbone

Evandro José Mulinari, PhD student and researcher at the São Carlos Institute of Physics of the University of São Paulo (IFSC / USP) provided the laccase from Thielavia terrestris (XP_003656170.1). The gene is inserted into the pETTrx-1a / LIC vector and transformed into E. coli Rosetta Gami 2 (DE3).

The gene was designed in such a way that, when expressed, the protein contains peptides with cleavage sites for VTE followed by thioredoxin and 6 histidines at the N-terminus, to make the purification easier of these proteins. The vector also contains genes of resistance to the antibiotic kanamycin and the expression is regulated by the Lac operon.

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