Team:USP-EEL-Brazil/Design

Step by Step

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 for the laccase expression constituted on the following desing:

PART NAME
INFORMATION

LacI Promoter

Obtained from the iGEM Registry: BBa_R0010 and found on the 2018 DNA Distribution Kit (kit plate 3, well 4G).

RBS

Sequence obtained from iGEM Registry: BBa_B0030 and synthesized with the gene.

Laccase from Pleurotus ostreatus - Lac PL

Laccase gene from Pleurotus ostreatus NRRL0366:3471 (Lac PL), was designed and synthesized by the team.

Laccase from Phoma sp. - Lac PL

Laccase gene from Phoma sp. UHH 5-1-03 (DSM 2245) (Lac PH), was designed and synthesized by the team.

Terminator

Found on the pSBIC3 backbone.

Also, our instructor Evandro Mulinari provided for us a laccase from Thielavia terrestris NRRL 8126 of his own design.

The gene was designed in such a way that, when expressed, the protein contains peptides with cleavage sites for TEV followed by thioredoxin and 6 histidines at the N-terminus, to make the protein purification easier.

Laccase Thielava terrestris (Lac TT):

pETTrx-1a / LIC vector:

This laccase (Lac TT) was inserted into the pETTrx-1a / LIC vector, witch contains resistance to kanamycin and has the expression regulated by the Lac operon. The transformation was done into E. coli Rosetta Gami 2 (DE3).

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