Part:BBa_K942004:Experience

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Applications of BBa_K942004

Our shuttle sequence can be quite useful to other iGEM teams and anyone with the objective of producing or comparing the expression of proteins in two different expression systems. Coupled with an codon optimization for Pichia p. and a strain capable of reading rare codons like Rosetta Gami DE3 pLys, Bl21 star, the outcome can be a simultanious expression in both organisms coming from one single genetic sequence.

User Reviews

UNIQ116361e841efcd74-partinfo-00000000-QINU UNIQ116361e841efcd74-partinfo-00000001-QINU

This construct was used for the expression of Der f 2 in Pichia pastoris. Also, thanks to its B.P.S. (bacterial promoter sequence) it is intended to be expressed in Escherichia coli.

We were able to successfully transform the construct into our cloning strain (TOP10 F); nevertheless, our expression strains (BL21(DE3) & JM109(DE3) strains) were not even able to be transformed, except from Rosetta gami (DE3) pLysS.

With our current evidence, and considering the fact that Der f 2 had been previously proposed to have antibacterial activity (Ichikawa S, 1998), we believe it is possible that BL21(DE3) and JM109(DE3) had been effectively transformed with the plasmid; but due to leaky expression of Der f 2, the cells might have been unable to grow any further and produce colonies.

This part uses a secretion signal for yeasts, and the protein produced was obtained by inducing Pichia Pastoris with .5% methanol.

We were able to se the expression of this protein on a SDS-PAGE after purifying it with a His-tag affinity column. Currently, we are trying different conditions to achieve the expression in BL21 Star.

The supernatant was obtained by centrifugation (10 ml at 3000 g, 5 min). After that, it was purified by the use of Maxi metal chelate spin clumns, from sartorius stedim biotech. Futhermore it was concentrated by using 3000 Dalton mwco ultracentrifuge filters. Obtaining a final concentration of .8 ug/ul in 500ul.

Tricine 16% gels were usedn order to see the protein by sds-page. Third lane = Derf (16KDa molecular weight) Fourth Lane = SeeBlue® Plus2 Pre-Stained