Difference between revisions of "Part:BBa K942004:Experience"

Revision as of 00:56, 29 September 2012

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_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

UNIQ1e5ceb9bd6869316-partinfo-00000000-QINU UNIQ1e5ceb9bd6869316-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. We believe that the fact that Rosetta gami had pLys to prevent leak expression and the other expression strains did not, leads us to the idea that there might be posibility of Der f 2 having antibacterial effects, as it is describe on the main page of this same part.

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.

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

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 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 successfully transformed the gene into our cloning strains, but curiously, our expression strains (DE3 strains) were not even able to be transformed, except from Rosettagami DE3 pLys. We believe that the fact that Rosettagami had pLys to prevent leak expression and the other expression strains did not, leads us to the idea that there might be posibility of Der f 2 having antibacterial effects, as it is describe on the main page of this same part.
+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. We believe that the fact that Rosetta gami had pLys to prevent leak expression and the other expression strains did not, leads us to the idea that there might be posibility of Der f 2 having antibacterial effects, as it is describe on the main page of this same part.
 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.