Difference between revisions of "Part:BBa K325219:ArabinosetoLight"

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(Protocol)
 
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'''Description'''<br>
 
'''Description'''<br>
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter.
 
D-Luciferin has to be added to obtain light output.
 
  
 +
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below.
  
'''Performance'''<br>
+
==Data==
 +
 
 +
[[Image:IntegratedactiPP+LRE.png|thumb|569px|center|'''Figure 1 - Transfer function of <partinfo>K325219</partinfo>.  The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. ''']]
 +
[[Image:MaximumlumPP+LRE.png|thumb|569px|center|'''Figure 2 - Maximum luminescence output of  <partinfo>K325219</partinfo> as a function of Arabinose concentration. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value. ''']]
 +
[[Image:TimePP+LRE.png|thumb|569px|center|'''Figure 3 - Evolution of luminescence with time at different Arabinose concentrations. The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.''']]
 
<center>
 
<center>
 
{|{{Table}}
 
{|{{Table}}
!Experiment<sup>1</sup>
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!Data
!Characteristic<sup>1</sup>
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!Notes
!Value<sup>1</sup>
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!Date Uploaded
 
|-
 
|-
|rowspan="3"|[[Part:BBa_F2620:Transfer Function|'''Transfer Function''']]
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|[[Media:BBa_K325219ArabinosetoLight.xls]]
|''Maximum Output''
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|Raw data from experiment
|6.6 [[PoPS]] cell<sup>-1</sup>
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|21/10/2010
|-
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|''Hill coefficient''
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|1.6
+
|-
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|[[Switch Point|''Switch Point'']]
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|1.5E-9 M [[3OC6HSL|3OC<sub>6</sub>HSL]], exogenous
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|-
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|[[Part:BBa_F2620:Response time|'''Response time:''']]
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|<1 min
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|-
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|rowspan="2"|[[Part:BBa_F2620:Specificity|'''Input compatibility''']]
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|''Strong response to''
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|[[3OC6HSL|3OC<sub>6</sub>HSL]], C<sub>6</sub>HSL , C<sub>7</sub>HSL, 3OC<sub>8</sub>HSL, C<sub>8</sub>HSL
+
|-
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|''Weak response to''
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|C<sub>4</sub>HSL, C<sub>10</sub>HSL, C<sub>12</sub>HSL
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|-
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|rowspan="2"|[[Part:BBa_F2620:Stability|'''Stability''']]
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|[[Genetic Stability|''Genetic Stability'']]<br>(Low/High Input)
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|>92/>56 generations
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|-
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|[[Performance Stability|''Performance Stability'']]<br>(Low/High Input)
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|>92/>56 generations
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|-
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|rowspan="4"|Demand
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|rowspan="1"|Internal Demand<br>(Low/High Input)
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|Not measured
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|-
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|rowspan="2"|[[Transcription Demand|''Transcriptional output demand:'']]<br>(Low/High Input)<br>Nt = length of downstream transcript in nucleotides
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|(0/6xNt) nucleotides cell<sup>-1</sup> s<sup>-1</sup>
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|-
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|(0/1.5E-1xNt) RNAP cell<sup>-1</sup>
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|-
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|[[Growth Rate|''Growth Rate'']]<br>(Low/High Input)
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|54/59 min Doubling time
+
 
|}
 
|}
 
</center>
 
</center>
<sup>1</sup>Measured by the [http://2010.igem.org/Team:Cambridge Cambridge iGEM team 2010]
+
 
 +
 
 +
</div>Measured by the [http://2010.igem.org/Team:Cambridge Cambridge iGEM team 2010]
 
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<div style="padding: 00px; width: 680px">
 
'''Compatibility'''<br>
 
'''Compatibility'''<br>
Line 66: Line 36:
 
'''References'''<br>
 
'''References'''<br>
 
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.
 
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.
</div>
+
 
 +
 
 
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.
 
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.
 +
 +
 
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.
 
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.

Latest revision as of 20:22, 27 October 2010

Input: L-Arabinose
Output: Light

pBad/araC
I0500
Luciferase/LRE
K325210
Cambridge-Eglowli.png

Part Main Page        Arabinose -> Light        Add Data       


Description

This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below.

Data

Figure 1 - Transfer function of BBa_K325219. The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value.
Figure 2 - Maximum luminescence output of BBa_K325219 as a function of Arabinose concentration. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.
Figure 3 - Evolution of luminescence with time at different Arabinose concentrations. The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.
Data Notes Date Uploaded
Media:BBa_K325219ArabinosetoLight.xls Raw data from experiment 21/10/2010


Measured by the [http://2010.igem.org/Team:Cambridge Cambridge iGEM team 2010]

Compatibility
Chassis: Device has been shown to work in Top 10 (Invitrogen)
Plasmids: Device has been shown to work on pSB1C3


References
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 [1]:] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,Life 61, 6-17.


[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html [2]:] T. Nakatsu et al. (2006) Structural Basis for the spectral difference in luciferase bioluminescence, Nature 440(16), 372-376.


[http://www.ncbi.nlm.nih.gov/pubmed/11457857 [3]:] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, The Journal of Biological Chemistry, 276(39), 36508-36513.