Regulatory

Part:BBa_K2656003

Designed by: Adrián Requena Gutiérrez   Group: iGEM18_Valencia_UPV   (2018-09-16)
Revision as of 18:59, 18 October 2021 by JeanHerdoiza (Talk | contribs)


Promoter HSL-mediated luxR

domestication.
Figure 1. DNA basic parts domestication. First construction refers to promoter GB domestication.


Part BBa_K2656003 is the promoter BBa_R0062 compatible with both Biobrick and [http://2018.igem.org/Team:Valencia_UPV/Design GoldenBraid 3.0] assembly methods. This promoter is positively regulated by LuxR together with Acyl-homoserine-lactone. It can be combined with other compatible parts from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Valencia UPV IGEM 2018 Printeria Collection] to assemble transcriptional units with the Golden Gate assembly protocol .

Ecuador 2021's characterization

We characterized this promoter using the composite part K3893028 that we built by assembling together

  • 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)

Device K3893028 allows us to measure GFP expression levels for different AHL induction concentrations.


domestication.
Figure 1. SBOL Visual representation of the device K3893028 from Ecuador 2021 Team


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.

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 𛼠𛼠= 8.11e8 molec min-1
Effective dissociation constant Kd Kd = 15.34 nM
Hill coefficient n = 0.958
Basal expression 𛽠= 0.1049
Dilution rate μ μ = 0.0115 min-1

Using this optimized parameter we obtain 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, and K2656114).




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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None