Difference between revisions of "Part:BBa K118025:Experience"

Latest revision as of 17:38, 26 September 2012

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Applications of BBa_K118025

Limonene synthase expression with pRARE2 in C41 DE3 E.coli that can be used to synthesize limonene terpenes.

User Reviews

UNIQd8540c452fc64554-partinfo-00000000-QINU

No review score entered. iGEM TU Munich 2012

Comment to 2011 British Columbia's review: We succesfully used the limonene-synthase which is included in this biobrick for expression in yeast. We could also detect the production of limonene in vitro and in vivo. Check out our Part BBa_K801060!!!

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•••••

British Columbia iGEM 2011

This composite part was expressed in our C41 E. coli that contained a pRARE plasmid encoding rare tRNA synthases. However, when we tried to express the limonene synthase in yeast, we were not able to obtain evidence for protein expression or function. So, we recommend that this part only be introduced to organisms capable of expressing foreign DNA.

UNIQd8540c452fc64554-partinfo-00000004-QINU

Characterization by British Columbia iGEM 2011

GC-MS analysis of Limonene Synthase function by British Columbia iGEM 2011

The limonene generator with lac promoter (BBa_K118025) includes the limonene generator (BBa_K118024) under the control of a lac promoter (BBa_J33207). This composite part's purpose is to express the limonene synthase (BBa_I742110), which is also available with a ribosome site (BBa_I742111).

Gas Chromatography Mass Spectrum of (+)-Limonene: We expressed the limonene generator with lac promoter (BBa_K118025) in C41 DE3 E. coli and lysed the cell culture to obtain unpurified protein extract. The crude extract was used in an in vitro enzymatic assay to produce limonene from geranyl diphosphate (GPP) substrate. The samples were prepared and analyzed by gas chromatography mass spectrometry. (A) The gas chromatography retention time of 9.9 minutes and (B) the mass spectrometry base peak at 93m/z (Figure B) are characteristic of limonene. (A) We analysed a positive limonene-containing control that yielded a strong peak as indicated by the black line as well as a negative control where no GPP was added to the assay, which yielded no peak as indicated by the red line. In comparison, our expressed limonene synthase that was incubated with GPP (indicated by the blue line) shows the indicative limonene peak at 9.9 minutes. (B) Cross reference with a compound library revealed that the limonene synthase sample's peak chromatography at 9.9 minutes (top panel) matches the library's d-limonene peak chromatography (bottom panel).

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 ===User Reviews===
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-<!-- Template for a user review
 {|width='80%' style='border:1px solid gray'
 |-
 |width='10%'|
 <partinfo>BBa_K118025 AddReview number</partinfo>
-<I>Username</I>
+<I>iGEM TU Munich 2012</I>
 |width='60%' valign='top'|
-Enter the review inofrmation here.
+Comment to 2011 British Columbia's review: We succesfully used the limonene-synthase which is included in this biobrick for expression in yeast. We could also detect the production of limonene in vitro and in vivo. Check out our Part <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K801060">BBa_K801060</a></html>!!!
 |};
 <!-- End of the user review template -->
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_K118025 AddReview 5</partinfo>
+<I>British Columbia iGEM 2011</I>
+|width='60%' valign='top'|
+This composite part was expressed in our C41 E. coli that contained a pRARE plasmid encoding rare tRNA synthases. However, when we tried to express the limonene synthase in yeast, we were not able to obtain evidence for protein expression or function. So, we recommend that this part only be introduced to organisms capable of expressing foreign DNA.
+|}
 <!-- DON'T DELETE --><partinfo>BBa_K118025 EndReviews</partinfo>
-==Characterization==
+==Characterization by British Columbia iGEM 2011==
-===GC-MS analysis of Limonene Synthase function===
+===GC-MS analysis of Limonene Synthase function by British Columbia iGEM 2011===
 The limonene generator with lac promoter (<partinfo>BBa_K118025</partinfo>) includes the limonene generator (<partinfo>BBa_K118024</partinfo>) under the control of a lac promoter (<partinfo>BBa_J33207</partinfo>). This composite part's purpose is to express the limonene synthase (<partinfo>BBa_I742110</partinfo>), which is also available with a ribosome site (<partinfo>BBa_I742111</partinfo>).
-[[Image:Ubcigem2011limonenegcms.jpg | frame | center | '''Gas Chromatography Mass Spectrum of (+)-Limonene:''' We expressed the limonene generator with lac promoter (<partinfo>BBa_K118025</partinfo>) in C41 DE3 ''E. coli'' and lysed the cell culture to obtain unpurified protein extract. The crude extract was used in an ''in vitro'' enzymatic assay to produce limonene from geranyl diphosphate (GPP). The samples were prepared and analyzed by gas chromatography mass spectrometry. At retention time 8.864 minutes, the base peak at 93m/z is characteristic of limonene.]]
+[[Image:Ubcigem2011limonenegcms.jpg | frame | center | '''Gas Chromatography Mass Spectrum of (+)-Limonene:''' We expressed the limonene generator with lac promoter (<partinfo>BBa_K118025</partinfo>) in C41 DE3 ''E. coli'' and lysed the cell culture to obtain unpurified protein extract. The crude extract was used in an ''in vitro'' enzymatic assay to produce limonene from geranyl diphosphate (GPP) substrate. The samples were prepared and analyzed by gas chromatography mass spectrometry. (A) The gas chromatography retention time of 9.9 minutes and (B) the mass spectrometry base peak at 93m/z (Figure B) are characteristic of limonene. (A) We analysed a positive limonene-containing control that yielded a strong peak as indicated by the black line as well as a negative control where no GPP was added to the assay, which yielded no peak as indicated by the red line. In comparison, our expressed limonene synthase that was incubated with GPP (indicated by the blue line) shows the indicative limonene peak at 9.9 minutes. (B) Cross reference with a compound library revealed that the limonene synthase sample's peak chromatography at 9.9 minutes (top panel) matches the library's d-limonene peak chromatography (bottom panel).]]
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