Difference between revisions of "Part:BBa K942004:Experience"
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===Applications of BBa_K942004=== | ===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 | + | 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=== | ===User Reviews=== | ||
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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''. | 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 | 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 | + | 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 | + | |
− | Third lane = | + | 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 | Fourth Lane = SeeBlue® Plus2 Pre-Stained | ||
[[Image:Tecmty_derfsds.jpg]] | [[Image:Tecmty_derfsds.jpg]] |
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
UNIQ3692a6eaba6ad58c-partinfo-00000000-QINU UNIQ3692a6eaba6ad58c-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