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

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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''.  
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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 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.  
  
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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.  
+
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.
  
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This part uses a secretion signal for yeasts, and the protein produced was obtained by inducing ''Pichia Pastoris'' with .5% methanol.  
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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 secreted to the medium
  
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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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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).
  
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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 columns, from sartorius stedim biotech.
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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.
 
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 were usedn order to see the protein by sds-page.
 
Tricine 16% gels were usedn order to see the protein by sds-page.
 
Third lane = Derf (16KDa molecular weight)
 
Third lane = Derf (16KDa molecular weight)
 
Fourth Lane = SeeBlue® Plus2 Pre-Stained
 
Fourth Lane = SeeBlue® Plus2 Pre-Stained
 +
 
[[Image:Tecmty_derfsds.jpg]]
 
[[Image:Tecmty_derfsds.jpg]]

Revision as of 03:43, 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 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

UNIQ3762d079c7010c7a-partinfo-00000000-QINU UNIQ3762d079c7010c7a-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. 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 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).

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

Tecmty derfsds.jpg