Difference between revisions of "Part:BBa K784001"
 
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<!-- Below contains the characterization added to this part by EPFL 2019 -->
 
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=='''Expression of CDO in OnePot PURE'''==
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=='''Expression of the gene XylE into CDO enzyme using the homemade cell-free system OnePot PURE - EPFL,2019'''==
  
  
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[[File:resultsOnePot.png|thumb|center|upright=3|SDS ]]
 
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[[File:T--EPFL--resultsOnePot.png]]
 
 
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'''Group: EPFL, 2019'''
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''' Group: ''' <html><a style="padding: 0px; margin: 0px;" href="https://2019.igem.org/Team:EPFL"> EPFL, 2019</a></html>
 
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'''Authors: Laura Kvedarauskaite, Konstantinos Ragios '''
 
 
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'''Hypothesis:'''
 
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'''Summary:''' We compared the amount of GFP expressed under a constitutive promoter (medium promoter, strong RBS) to T7 expression. Some proteins fold better under constitutive promoters; however, nobody had yet directly compared the amount of protein produced between constitutive vs. T7 expression.  
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Our goal was to express the gene XylE, into the enzyme Catechol 2,3-dioxygenase (CDO), using the cell free system OnePot PURE. CDO reacts with the substrate catechol to transform into another product, which causes a color change from transparent to yellow. For the purpose of our project we needed to induce this color change in order to create a signal.
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'''Methods'''
 
'''Methods'''
 
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BioBricks were transformed and expressed in E. coli (BL21). BL21 cells were cultured to an OD600=0.6 and 100 uL of culture was transferred into a 96 well plate. Colonies were transfered in quadruplicate. The fluorescence intensity of GFP was measured with a multi-mode microplate reader. The iGEM standardized fluorescence protocol was used for fluorescence measurement standardization (https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-6zrhf56).
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To obtain this part, we started from the part BBa_J33204 to which we added a T7 promoter and T7 terminator.
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To express this gene into the cell free system OnePot PURE, we followed the <html><a style="padding: 0px; margin: 0px;" href="https://www.protocols.io/view/cdo-expression-into-onepot-pure-7nhhmb6"> protocol</a></html> we created . To assert the validity of the results, we performed two controls next to the reaction. The first control expressed XylE without catechol, and the second one had catechol but no XylE template. We incubated the reactions at 37°C and took pictures regularly to see how the color was changing throughout the reaction.
  
 
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'''Results:'''
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'''Results'''  
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<br/> This picture shows the evolution of the reaction of CDO with catechol as well as the state of the controls. The first column are the pictures of the reaction. The second and third columns are controls, respectively, one with XylE template but without catechol, the other with catechol but without XylE template. A color change is observed in the reaction; after 30 minutes of incubation the color starts to become yellow and after 1 hour the color is bright and distinguishes clearly from the controls. Both controls remain transparent throughout the runtime.
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We found that the T7 promoter produced about 2.6 times as much fluorescent signal as the constitutive PR4 promoter, indicating that T7 is much more efficient at producing GFP ('''Fig. 5'''). Interestingly, the production of GFP under PR4 did not increase beyond the level observed at 4 hours after inoculation. It seems that PR4 leads to an initial production of protein; however, after the initial expression the promoter seems to be shut off.
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[[File:T--Queens_Canada--Bronze_PR4vsT7.png|thumb|600px|left|<b>Figure 5.</b> Fluorescent intensity of BL21 expressing GFP under T7 (blue), and PR4 (Green). The T7 promoter lead to about 2.6 as much protein being produced over a 16 hour timescale. Fluorescent intensity was determined using the fluorescein standard and logarithmic curves.  
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'''Promoter and RBS:'''<br/>
 
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PR4: medium Promoter (J23110) strong RBS (B0034)<br/>
 
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T7: T7 Promoter (BBa_I746909) <br/>
 
  
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{|cellpadding="10" cellspacing="0" border="1"
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<html>
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|sample
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<figure style="text-align:center;">
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|PR4
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                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/9/9d/T--EPFL--Detection_cdo_result.jpg" alt="control">
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|'''T7'''
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                <figcaption><b>Figure 1:</b> CDO expression.</figcaption>
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              </figure>
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|<span style="color:green;">Fluorescence 4 hrs (a.u.)</span>
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</html>
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|2.24E+05
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'''Conclusion'''
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|'''3.09E+05'''
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<br/>The gene XylE with a T7 promoter was successfully expressed in the cell free system OnePot PURE into its corresponding enzyme, CDO. The reaction between CDO and catechol induces a color change from transparent to yellow which was clearly observed after 1 hour of incubation at 37°C.
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|<span style="color:green;">Fluorescence 16 hrs (a.u.)</span>
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|2.48E+05
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Latest revision as of 23:19, 21 October 2019

pT7_RBS_xyIE reporter gene

This part's purpose is to testify to the presence of the T7 RNA Polymerase by the xyIE reporter gene from iGEM2006_Edinburgh. It consists of an RNA T7 promoter, an RBS, a reporter gene- xyIE, and a T7 terminator. The assay for xyIE is very simple, refer to the xyIE basic part for instructions.
The xyIE+RBS part is between XhoI and XmaI restriction sites, therefore can be excised by these enzymes.


Expression of the gene XylE into CDO enzyme using the homemade cell-free system OnePot PURE - EPFL,2019


Group: EPFL, 2019

Hypothesis:
Our goal was to express the gene XylE, into the enzyme Catechol 2,3-dioxygenase (CDO), using the cell free system OnePot PURE. CDO reacts with the substrate catechol to transform into another product, which causes a color change from transparent to yellow. For the purpose of our project we needed to induce this color change in order to create a signal.



Methods
To obtain this part, we started from the part BBa_J33204 to which we added a T7 promoter and T7 terminator. To express this gene into the cell free system OnePot PURE, we followed the protocol we created . To assert the validity of the results, we performed two controls next to the reaction. The first control expressed XylE without catechol, and the second one had catechol but no XylE template. We incubated the reactions at 37°C and took pictures regularly to see how the color was changing throughout the reaction.


Results:
This picture shows the evolution of the reaction of CDO with catechol as well as the state of the controls. The first column are the pictures of the reaction. The second and third columns are controls, respectively, one with XylE template but without catechol, the other with catechol but without XylE template. A color change is observed in the reaction; after 30 minutes of incubation the color starts to become yellow and after 1 hour the color is bright and distinguishes clearly from the controls. Both controls remain transparent throughout the runtime.


control
Figure 1: CDO expression.
Conclusion
The gene XylE with a T7 promoter was successfully expressed in the cell free system OnePot PURE into its corresponding enzyme, CDO. The reaction between CDO and catechol induces a color change from transparent to yellow which was clearly observed after 1 hour of incubation at 37°C.




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 368
    Illegal NgoMIV site found at 540
    Illegal AgeI site found at 891
  • 1000
    COMPATIBLE WITH RFC[1000]