Composite

Part:BBa_K3893028

Designed by: Jean Pierre Herdoiza   Group: iGEM21_Ecuador   (2021-10-16)

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 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 Valencia_UPV 2018 Team. Check our experimental page for more details, here you can get the raw data.

Using the same 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]


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Categories
Parameters
None