Difference between revisions of "Part:BBa E0030:Experience"

Revision as of 01:46, 4 October 2012

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Applications of BBa_E0030

Characterization by British Columbia iGEM 2012

YFP fluorescence output by British Columbia iGEM 2012

The strong constitutive promoter-EYFP generator (BBa_K804001) includes an enhanced yellow fluorescence protein (BBa_E0030) under the control of a constitutive pTet promoter (BBa_J23118). This composite part's purpose is to constitutively express the YFP(BBa_E0030), which is also available with a strong ribosome binding site (BBa_B0034).

Fluorescence Output of EYFP under a pTet constituitive promoter in co-culture and mono-culture : MetA- auxotroph is transformed with the EYFP construct (BBa_K804001) under the constitutive pTet promoter (BBa_J23118). We co-cultured this with TyrA- auxotrophs containing a ECFP construct (BBa_K804000)under the same constitutive pTet promoter; as well as with TrpA- auxotrophs containing a RFP construct (BBa_K081012) under the same constitutive pTet promoter. Fluorescence output of EYFP for the three member co-culture, pairwise co-culture, and mono-culture with MetA- auxotrophs are analyzed using a plate reader which measures emission of YFP at a wavelength of 527nm when excited at 514nm.

User Reviews

UNIQa95af902adb86be2-partinfo-00000009-QINU UNIQa95af902adb86be2-partinfo-0000000A-QINU

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 ===YFP fluorescence output by British Columbia iGEM 2012===
-The strong constitutive promoter-EYFP generator  includes an enhanced yellow fluorescence protein  (<partinfo>BBa_E0030</partinfo>) under the control of a constitutive pTet promoter (<partinfo>BBa_J23118</partinfo>). This composite part's purpose is to constituitively express the YFP(<partinfo>BBa_E0030</partinfo>), which is also available with a strong ribosome binding site (<partinfo>BBa_B0034</partinfo>).
+The strong constitutive promoter-EYFP generator (<partinfo>BBa_K804001</partinfo>) includes an enhanced yellow fluorescence protein  (<partinfo>BBa_E0030</partinfo>) under the control of a constitutive pTet promoter (<partinfo>BBa_J23118</partinfo>). This composite part's purpose is to constitutively express the YFP(<partinfo>BBa_E0030</partinfo>), which is also available with a strong ribosome binding site (<partinfo>BBa_B0034</partinfo>).
-<p align=center>https://static.igem.org/mediawiki/2012/2/29/UBCYFPCultures.png</p> '''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).]]
+<p align=center>https://static.igem.org/mediawiki/2012/2/29/UBCYFPCultures.png</p> '''Fluorescence Output of EYFP under a pTet constituitive promoter in co-culture and mono-culture :''' MetA- auxotroph is transformed with the EYFP construct (<partinfo>BBa_K804001</partinfo>) under the constitutive pTet promoter (<partinfo>BBa_J23118</partinfo>). We co-cultured this with TyrA- auxotrophs containing a ECFP construct (<partinfo>BBa_K804000</partinfo>)under the same constitutive pTet promoter; as well as with TrpA- auxotrophs containing a RFP construct (<partinfo>BBa_K081012</partinfo>) under the same constitutive pTet promoter. Fluorescence output of EYFP for the three member co-culture, pairwise co-culture, and mono-culture with MetA- auxotrophs are analyzed using a plate reader which measures emission of YFP at a wavelength of 527nm when excited at 514nm.
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