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

Latest revision as of 22:52, 30 September 2012

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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 pastoris and a strain capable of reading rare codons like Rosetta Gami DE3 pLysS, Bl21 star (DE3), the outcome can be a simultaneous expression in both organisms coming from one single genetic sequence.

User Reviews

UNIQe38206c27edefe6a-partinfo-00000000-QINU UNIQe38206c27edefe6a-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 achieved expression of the protein in P. pastoris cultures grown in 0.5% methanol and enough aeration. Because this part uses a secretion signal for yeasts, the protein was mainly localized in the growth medium. After being harvested, the cultures were centrifuged (5000g , 4º C, 5 min), and the supernatant was collected and purified using Vivapure Metal Chelate Maxi spin columns (Sartorius Stedim Biotech). Futhermore, the samples were concentrated with 3 kDa ultracentrifuge filters, obtaining a final concentration of 0.37 ug/uL in 500uL.Finally, Der f 2 was detected in a SDS-PAGE gel (Tris-Tricine 16%, 90 V, 2.5 h run-time) with silver staining.

We are currently trying different conditions to achieve the expression in BL21 Star (DE3), JM109 (DE3) and Rosetta gami pLysS.

Tricine 16% gels loaded with purified samples from transformed P.pastoris cultures. The band representing Der f 2 was visualized after silver staining.

Third lane = Der f 2 (16 kDa olecular weight)

Fourth Lane = SeeBlue® Plus2 Pre-Stained

@@ Line 5: / Line 5: @@
 ===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.
+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 pastoris'' and a strain capable of reading rare codons like Rosetta Gami DE3 pLysS, Bl21 star (DE3), the outcome can be a simultaneous expression in both organisms coming from one single genetic sequence.
 ===User Reviews===
@@ Line 23: / Line 23: @@
 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. Rosetta gami pLysS, on the other hand, has the capacity to suppress expression from the T7 promoter; and this may be the reason for its survival and growth.
+We achieved expression of the protein in ''P. pastoris'' cultures grown in 0.5% methanol and enough aeration. Because this part uses a secretion signal for yeasts, the protein was mainly localized in the growth medium. After being harvested, the cultures were centrifuged (5000g , 4º C, 5 min), and the supernatant was collected and purified using
+Vivapure Metal Chelate Maxi spin columns (Sartorius Stedim Biotech). Futhermore, the samples were concentrated with 3 kDa ultracentrifuge filters, obtaining a final concentration of 0.37 ug/uL in 500uL.Finally, Der f 2 was detected in a SDS-PAGE gel (Tris-Tricine 16%, 90 V, 2.5 h run-time) with silver staining.
-We achieved expression of the protein in ''P. pastoris'' cultures grown in 0.5% methanol and enough aeration. Because this part uses a secretion signal for yeasts, the protein was secreted to the medium
- 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).
+We are currently trying different conditions to achieve the expression in BL21 Star (DE3), JM109 (DE3) and Rosetta gami pLysS.
-After that, it was purified by the use of Maxi metal chelate spin columns, 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.
-We were able to detect 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.
+Tricine 16% gels loaded with purified samples from transformed ''P.pastoris'' cultures. The band representing Der f 2 was visualized after silver staining.
+Third lane = Der f 2 (16 kDa olecular weight)
-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
 [[Image:Tecmty_derfsds.jpg]]