Difference between revisions of "Part:BBa K3893028"
 
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This device was used to characterize the pLux promoter. It is an AHL induced transcriptional unit to express GFP together with a TU for the constitutive expression of LuxR. It was assembled with a one-pot Level 2 Golden Gate reaction using BsmBI type IIS endonuclease.
 
This device was used to characterize the pLux promoter. It is an AHL induced transcriptional unit to express GFP together with a TU for the constitutive expression of LuxR. It was assembled with a one-pot Level 2 Golden Gate reaction using BsmBI type IIS endonuclease.
  
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This device is composed of the following standardized composite parts from Valencia_UPV 2018 iGEM Team [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]:  
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This composite part, together with [https://parts.igem.org/Part:BBa_K3893029 K3893029], is an intermediate composite part to assemble the release system device ([https://parts.igem.org/Part:BBa_K3893030 K3893030]) of our Agrobactory 593. It includes a TU that expresses GFP under the control of the pLux promoter and a TU that expresses LuxR constitutively. It is the first half of our population oscillator release device.
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This transcriptional unit is composed of the following standardized composite parts from Valencia_UPV 2018 iGEM Team [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]:  
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<html>
 
<html>
 
<ul>
 
<ul>
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===Usage and Biology===
 
===Usage and Biology===
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This composite part, together with [https://parts.igem.org/Part:BBa_K3893029 K3893029], is an intermediate composite part to assemble the release system device ([https://parts.igem.org/Part:BBa_K3893030 K3893030]) of our Agrobactory 593. It includes a TU that expresses GFP under the control of the pLux promoter and a TU that expresses LuxR constitutively. It is the first half of our population oscillator release device.
  
 
==Characterization==
 
==Characterization==
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Following a standard experimental procedure and the IGEM 2018 Interlab calibration for Particles and MEFL we obtained the temporal behavior of the device for different inducer concentrations.  
 
Following a standard experimental procedure and the IGEM 2018 Interlab calibration for Particles and MEFL we obtained the temporal behavior of the device for different inducer concentrations.  
  
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[[File:T--Ecuador--K3893028_time_response.png|600px|thumb|center|alt=domestication.|Experimental reuslts for characterization of pLux promoter.]]
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[[File:T--Ecuador--K3893028_time_response.png|600px|thumb|center|alt=domestication.|Experimental results for characterization of pLux promoter.]]
  
 
[[File:T--Ecuador--K3893028_static_response.png|600px|thumb|center|alt=domestication.|Final GFP expression levels for different AHL inducer concentrations.]]
 
[[File:T--Ecuador--K3893028_static_response.png|600px|thumb|center|alt=domestication.|Final GFP expression levels for different AHL inducer concentrations.]]
  
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<!-- <html>
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<table style="width:50%">
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We used this part to characterize the Golden Braid version ([https://parts.igem.org/Part:BBa_K2656003 K2656003]) of pLux promoter ([https://parts.igem.org/Part:BBa_R0062 R0062]) made by [http://2018.igem.org/Team:Valencia_UPV Valencia_UPV 2018 Team]. Check our experimental page for more details,  [https://2021.igem.org/File:T--Ecuador--Exp_K3893028.xlsx here you can get the raw data.]
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              <tr>
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                <th><p>Experiment parameters</p></th>
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Using the same [http://2018.igem.org/Team:Valencia_UPV/Experiments#exp_protocol experimental protocol as Valencia_UPV team ], we have obtained the parameters for the following model:
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                <th><p>Description</p></th>
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              </tr>
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[[File:T--Ecuador_UPV--Model_pLux_Charact_eq.png|300px|thumb|center|alt=equation|]]
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              <tr>
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                <td><p>Time</p></td>
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{|class='wikitable'
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                <td><p>06:00:00 (HH:MM:SS). Measurement interval: 05:00 (MM:SS)</p></td>
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|colspan=4|Table 1. Parameters obtained from part K3893028 corresponding to K2656003.
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              </tr>
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              <tr>
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|'''Parameter'''
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                <td><p>Number of samples</p></td>
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|'''Value'''
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                <td><p>8 samples for each TU colony</p></td>
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              </tr>
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|Effective translation rate 𝛼
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              <tr>
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|𝛼 = 8.11e8 molec min-1
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                <td><p>Number of medium samples</p></td>
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                <td><p>8 samples of M9 medium</p></td>
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|Effective dissociation constant Kd
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              </tr>
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|Kd = 15.34 nM
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              <tr>
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                <td><p>Temperature</p></td>
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|Hill coefficient
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                <td><p>37 ºC</p></td>
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|n = 0.958
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              </tr>
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              <tr>
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|Basal expression
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                <td><p>Shake</p></td>
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|𝛽 = 0.1049
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                <td><p>Double Orbital. Continuously</p></td>
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              </tr>
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|Dilution rate μ
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                <tr>
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| μ = 0.0115 min-1
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                <td><p>Absorbance. Optical Density (OD) measure</p></td>
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|}
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                <td><p>Wavelenght at 600 nm emission</p></td>
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              </tr>
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Using this optimized parameter we obtained the simulated hill function corresponding to the previous equation and compared it with the experimental data obtained (for steady-state, this is after reaching equilibrium).
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              <tr>
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                <td><p>Excitacion wavelength</p></td>
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[[File:T--Ecuador_UPV--Model_pLux_Charact.png|600px|thumb|center|alt=Data.|Experimental data and simulation to characterize the pLux promoter]]
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                <td><p>Wavelenght at 485 nm</p></td>
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              </tr>
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Note: This characterization data for part R0062 ([https://parts.igem.org/Part:BBa_K2656003 K2656003]) needs to be interpreted in the context of the transcriptional units from where the experimental data was taken ([https://parts.igem.org/Part:BBa_K3893028 K3893028], [https://parts.igem.org/Part:BBa_K2656122 K2656122], [https://parts.igem.org/Part:BBa_K2656114 K2656114).]
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              <tr>
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                <td><p>Emission wavelength</p></td>
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                <td><p>Wavelenght at 528 nm</p></td>
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              </tr>
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              <tr>
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                <td><p>Gain (G)</p></td>
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                <td><p>60</p></td>
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              </tr>
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          </table></html> -->
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Latest revision as of 18:45, 19 October 2021

AHL induced expression of GFP (including luxR constitutive expression)

domestication.
Figure 1. SBOL Visual representation of the device

This device was used to characterize the pLux promoter. It is an AHL induced transcriptional unit to express GFP together with a TU for the constitutive expression of LuxR. It was assembled with a one-pot Level 2 Golden Gate reaction using BsmBI type IIS endonuclease.

This device is composed of the following standardized composite parts from Valencia_UPV 2018 iGEM Team [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]:

  • BBa_K2656122: a Level 1 transcriptional unit expresing GFP under the control of the pux promoter (Golden Braid compatible)
  • BBa_K2656114: a Level 1 transcriptional unit constitutively expressing luxR gene (Golden Braid compatible)

Usage and Biology

This composite part, together with K3893029, is an intermediate composite part to assemble the release system device (K3893030) of our Agrobactory 593. It includes a TU that expresses GFP under the control of the pLux promoter and a TU that expresses LuxR constitutively. It is the first half of our population oscillator release device.

Characterization

Following a standard experimental procedure and the IGEM 2018 Interlab calibration for Particles and MEFL we obtained the temporal behavior of the device for different inducer concentrations.

domestication.
Experimental results for characterization of pLux promoter.
domestication.
Final GFP expression levels for different AHL inducer concentrations.


We used this part to characterize the Golden Braid version (K2656003) of pLux promoter (R0062) made by [http://2018.igem.org/Team:Valencia_UPV Valencia_UPV 2018 Team]. Check our experimental page for more details, here you can get the raw data.

Using the same [http://2018.igem.org/Team:Valencia_UPV/Experiments#exp_protocol experimental protocol as Valencia_UPV team ], we have obtained the parameters for the following model:

equation
Table 1. Parameters obtained from part K3893028 corresponding to K2656003.
Parameter Value
Effective translation rate 𝛼 𝛼 = 8.11e8 molec min-1
Effective dissociation constant Kd Kd = 15.34 nM
Hill coefficient n = 0.958
Basal expression 𝛽 = 0.1049
Dilution rate μ μ = 0.0115 min-1

Using this optimized parameter we obtained the simulated hill function corresponding to the previous equation and compared it with the experimental data obtained (for steady-state, this is after reaching equilibrium).

Data.
Experimental data and simulation to characterize the pLux promoter

Note: This characterization data for part R0062 (K2656003) needs to be interpreted in the context of the transcriptional units from where the experimental data was taken (K3893028, K2656122, K2656114).



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 956
    Illegal NheI site found at 979
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]