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

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===Applications of BBa_K942004===
 
===Applications of BBa_K942004===
  
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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.
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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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<!-- DON'T DELETE --><partinfo>BBa_K942004 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K942004 EndReviews</partinfo>
  
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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''.
  
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This protein was expressed in Pichia pastoris. Thanks to its B.P.S. it was also planned to be expressed in Escherichia coli. We succesfully 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.
 
  
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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.
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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
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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.  
  
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This part uses a secretion signal for yeasts, and the protein produced was obtained by inducing Pichia Pastoris wih .5% methanol.
 
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The supernatant was obtained by centrifugation (10 ml at 3000 g, 5 min).
 
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After that, it was purified by the use of Maxi metal chelate spin clumns, from sartorius stedim biotech.
 
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Futhermore it was concentrated by using 3000 Dalton mwco ultracentrifuge filters. Obtaining a final concentration of .8 ug/ul in 500ul.
 
  
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Tricine 16% gels were usedn order to see the protein by sds-page.
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We are currently trying different conditions to achieve the expression in BL21 Star (DE3), JM109 (DE3) and Rosetta gami pLysS.
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Third lane = Derf (16KDa molecular weight)
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Fourth Lane = SeeBlue® Plus2 Pre-Stained Standard
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Tricine 16% gels loaded with purified samples from transformed ''P.pastoris'' cultures. The band representing Der f 2 was visualized after silver staining.
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Third lane = Der f 2 (16 kDa olecular weight)
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Fourth Lane = SeeBlue® Plus2 Pre-Stained
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[[Image:Tecmty_derfsds.jpg]]
 
[[Image:Tecmty_derfsds.jpg]]

Latest revision as of 22:52, 30 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 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

UNIQ651e38641ee7da79-partinfo-00000000-QINU UNIQ651e38641ee7da79-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

Tecmty derfsds.jpg