Difference between revisions of "Part:BBa K3893028"

(→‎Characterization)
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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.  
 
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.  
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Characterization of this part was performed with the transcriptional unit [https://parts.igem.org/Part:BBa_K2656101 BBa_K2656101 ], which was used in a comparative RBS expression experiment with composite parts [https://parts.igem.org/Part:BBa_K2656101 BBa_K2656101] and [https://parts.igem.org/Part:BBa_K2656102 BBa_K2656102].
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They all were assembled in a Golden Braid alpha1 plasmid using the same promoter, CDS and terminator:
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<html>
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<ul>
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<li></html>Promoter [https://parts.igem.org/Part:BBa_K2656004 BBa_K2656004]: the [https://parts.igem.org/Part:BBa_J23106 J23106] promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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<li></html>CDS [https://parts.igem.org/Part:BBa_K2656013 BBa_K2656013]: the [https://parts.igem.org/Part:BBa_K2656009 BBa_K2656009] sfGFP sequence in its Golden Braid standardized version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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<li></html>Terminator [https://parts.igem.org/Part:BBa_K2656026 BBa_K2656026]: the [https://parts.igem.org/Part:BBa_B0015 B0015] transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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</ul>
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</html>
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By using this [http://2018.igem.org/Team:Valencia_UPV/Experiments#exp_protocol experimental protocol], we have obtained the parameters to valide our  [http://2018.igem.org/Team:Valencia_UPV/Modeling#models constitutive model]and rationale choose its [http://2018.igem.org/Team:Valencia_UPV/Modeling#optimization optimization values] based on each RBS tested.
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[[File:T--Valencia_UPV--optimization_exp1_RBS_graphUPV2018.png|900px|thumb|none|alt=RBS experiment 1.|Figure 1. RBS expression experiment with K2656009, K26560010 and K2656011 RBS basic parts]]
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{|class='wikitable'
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|colspan=4|Table 1. Optimized parameters for the BBa_K2656009 RBS.
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|-
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|'''Parameter'''
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|'''Value'''
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|-
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|Translation rate p
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|p = 0.082 min-1
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|-
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|Dilution rate μ
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| μ = 0.01631 min-1
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|}
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We have also calculated the relative force between the different RBS, taking [https://parts.igem.org/Part:BBa_K2656009 BBa_K2656009 strong RBS] as a reference. It has been defined as the quotient between the values of the protein in equilibrium of the results of the simulation of one RBS and another reference RBS. Likewise, a ratio between p parameters of the different RBS parts and p parameter of the reference RBS has been calculated.
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{|class='wikitable'
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|colspan=4|Table 2. BBa_K2656008 (GB BBa_B0032 RBS) relative strength and p ratio.
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|-
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|'''Parameter'''
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|'''Value'''
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|-
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|Relative strength
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|0.371
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|-
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|p parameter ratio (pRBS/pref)
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|0.398
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|}
  
  

Revision as of 18:19, 18 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.


Characterization of this part was performed with the transcriptional unit BBa_K2656101 , which was used in a comparative RBS expression experiment with composite parts BBa_K2656101 and BBa_K2656102. They all were assembled in a Golden Braid alpha1 plasmid using the same promoter, CDS and terminator:

  • Promoter BBa_K2656004: the J23106 promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
  • CDS BBa_K2656013: the BBa_K2656009 sfGFP sequence in its Golden Braid standardized version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
  • Terminator BBa_K2656026: the B0015 transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
By using this [http://2018.igem.org/Team:Valencia_UPV/Experiments#exp_protocol experimental protocol], we have obtained the parameters to valide our [http://2018.igem.org/Team:Valencia_UPV/Modeling#models constitutive model]and rationale choose its [http://2018.igem.org/Team:Valencia_UPV/Modeling#optimization optimization values] based on each RBS tested.

RBS experiment 1.
Figure 1. RBS expression experiment with K2656009, K26560010 and K2656011 RBS basic parts


Table 1. Optimized parameters for the BBa_K2656009 RBS.
Parameter Value
Translation rate p p = 0.082 min-1
Dilution rate μ μ = 0.01631 min-1

We have also calculated the relative force between the different RBS, taking BBa_K2656009 strong RBS as a reference. It has been defined as the quotient between the values of the protein in equilibrium of the results of the simulation of one RBS and another reference RBS. Likewise, a ratio between p parameters of the different RBS parts and p parameter of the reference RBS has been calculated.

Table 2. BBa_K2656008 (GB BBa_B0032 RBS) relative strength and p ratio.
Parameter Value
Relative strength 0.371
p parameter ratio (pRBS/pref) 0.398



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]