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

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(→‎YFP fluorescence expression, under the control of PCMV promoter by BIOSINT_Mexico iGEM 2014)
 
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===Applications of BBa_E0030===
 
===Applications of BBa_E0030===
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==Characterization by BIOSINT_Mexico iGEM 2014==
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===YFP fluorescence expression, under the control of PCMV promoter by BIOSINT_Mexico iGEM 2014===
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As a part of the project from 2014_BIOSINT_Mexico , we fused the promoter PCMV (BBa_K747096) to a YFP reporter (BBa_E0030).
 +
 +
In order to measure its expression, we culture transformed E.coli in agar plates and measured the fluorescense intensity every 45 minutes. After that, we obtained the following data. [Fig.1]
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[[File:6res.png|150px|thumb|left|'''Figure 1''' YFP intensity every 45 minutes.]]
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We made three repetitions, so in order to model the equation, we obtained the mean measure, therefore:
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::::::[[File:2res.png|300px|thumb|left|'''Figure 2''' Media YFP intensity every 45 minutes.]]
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 +
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We analysed the data and using the Wolfram Mathematica software, we obtained the following equation that fits the data.
 +
If we plot this equation, we obtain:
 +
 +
 +
[[File:Model1res.png|400px|center|]]
 +
 +
Where we can see that the intensity of YFP grows directly proportional to the time and concentration of the molecule.
 +
 +
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::::::[[File:1res.png|600px|left|center|'''Figure 3''' Intensity YFP vs time.]]
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<br style="clear:both;"/>
  
 
==Characterization by British Columbia iGEM 2012==
 
==Characterization by British Columbia iGEM 2012==
  
 
===YFP fluorescence output by British Columbia iGEM 2012===
 
===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>).
  
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[[File: UBCYFPCultures.png|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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<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 with MetA- auxotroph grown over time :''' 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. These cells were grown in minimal media spiked with 10^-3M amino acids (methionine, tryptophan, and tyrosine) . 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.
  
 
<br style="clear:both;"/>
 
<br style="clear:both;"/>
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<!-- DON'T DELETE --><partinfo>BBa_E0030 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_E0030 EndReviews</partinfo>
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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_E0030 AddReview 5</partinfo>
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<I>2012 iGEM UIUC</I>
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|width='60%' valign='top'|
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The part worked as expected. The way we quantified it through a plate reader with emmission wavelengths. <br/><br/> Excitation: 515nm<br/>
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Emission: 528nm<br/><br/>
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We used this part for this project, data collected can be found at http://2012.igem.org/Team:UIUC-Illinois/Results.
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|};

Latest revision as of 02:01, 2 November 2014

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_E0030

Characterization by BIOSINT_Mexico iGEM 2014

YFP fluorescence expression, under the control of PCMV promoter by BIOSINT_Mexico iGEM 2014

As a part of the project from 2014_BIOSINT_Mexico , we fused the promoter PCMV (BBa_K747096) to a YFP reporter (BBa_E0030).

In order to measure its expression, we culture transformed E.coli in agar plates and measured the fluorescense intensity every 45 minutes. After that, we obtained the following data. [Fig.1]

Figure 1 YFP intensity every 45 minutes.

We made three repetitions, so in order to model the equation, we obtained the mean measure, therefore:

Figure 2 Media YFP intensity every 45 minutes.


We analysed the data and using the Wolfram Mathematica software, we obtained the following equation that fits the data. If we plot this equation, we obtain:


Model1res.png

Where we can see that the intensity of YFP grows directly proportional to the time and concentration of the molecule.


Figure 3 Intensity YFP vs time.


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).

UBCYFPCultures.png

Fluorescence Output of EYFP under a pTet constituitive promoter in co-culture and mono-culture with MetA- auxotroph grown over time : 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. These cells were grown in minimal media spiked with 10^-3M amino acids (methionine, tryptophan, and tyrosine) . 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

UNIQ6dd18d2e5341c618-partinfo-00000009-QINU UNIQ6dd18d2e5341c618-partinfo-0000000A-QINU

•••••

2012 iGEM UIUC

The part worked as expected. The way we quantified it through a plate reader with emmission wavelengths.

Excitation: 515nm
Emission: 528nm

We used this part for this project, data collected can be found at http://2012.igem.org/Team:UIUC-Illinois/Results.

;