Difference between revisions of "Part:BBa K3893028"

Revision as of 18:59, 18 October 2021

AHL induced expression of GFP (including luxR constitutive expression)

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.

Experimental results for characterization of pLux promoter.

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:

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 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).

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
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]

@@ Line 31: / Line 31: @@
 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.
-By 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:
+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:
 [[File:T--Ecuador_UPV--Model_pLux_Charact_eq.png|300px|thumb|center|alt=equation|]]
@@ Line 54: / Line 54: @@
 |-
 |Dilution rate μ
-| μ = d_2 = 0.0115 min-1
+| μ =  0.0115 min-1
 |}