Composite

Part:BBa_K575029

Designed by: Kristin Palarz   Group: iGEM11_Northwestern   (2011-09-22)
Revision as of 13:31, 21 October 2019 by Tkm (Talk | contribs) (→‎Characterization TokyoTech 2019)

LasR/PAI1 Inducible Promoter + RBS (B0034) + GFP, Constitutive Promoter + RBS (B0034) + LasR

Continuous expression of LasR (with RBS B0034), coupled with a LasR/PAI1 (3-oxo-C12-HSL) inducible promoter, RBS (Part B0034), and a GFP reporter.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 58
    Illegal NheI site found at 918
    Illegal NheI site found at 941
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1294
    Illegal AgeI site found at 1491
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 827


Characterization TokyoTech 2019

Author:iGEM TokyoTech2019 (https://2019.igem.org/Team:TokyoTech)

Overview:

Characterization is one of major requirement to test the validity and usability of the biobricks have already been in the registry. Our team selected two promoters to characterize: BBa K575029, which is LasR (with RBS B0034), coupled with a LasR/PAI1 (AHL, 3-oxo-C12-HSL) inducible promoter, RBS (Part B0034), and a GFP reporter. These sequences are working in E.coli. To quantify the activation of LasR/PAI1 inducible promoter, we are using Green Fluorescent Protein (GFP) and plotting fluorescence intensity and optical density every 15 minutes at different concentrations.

Experimental Procedure：[

・Amplify the part (BBa K575029) from iGEM 2019 Distribution Kit

・Transform into bacteria (DH5α)

・Plate the bacteria in Chloramphenicol-resistant plate


Data Analysis：

When successful colonies start growing, data collection can proceed.

１．Select a colony and inoculate it into 5mL of LB media with Chloramphenicol.

２．Starting from hour 0, pipette 100 uL of the liquid inoculate into a 96-well plate.

　　a. Leave the liquid inoculate in the incubator, shaking. b. Every 15 minutes, take out another 100uL and put it into the adjacent well and then store the well plate in 4°C. 　　i. 1st Row: After 0 minutes 　ii. 2nd Row: After 15 minutes 　iii. 3rd Row: After 30 minutes etc. ３．After collecting the samples for between 4 hours, run it on the microplate reader. 　　a. Collect the OD 660 by microplate reader b. Collect the fluorescence intensity from GFP

Result： It was confirmed that the fluorescence intensity of each E. coli increased with increasing PAI2 (AHL) concentration. In this assay for BBa K575029, the threshold value for the AHL addition amount was between 56 µM and 112 µM.

Note: The fluorescence intensity did not increase under the addition of 225µM PAI2. From this, there is a threshold for the induction of PAI2 on pLas, which is considered correct because LasR is driven to expression by a constitutive promoter and its expression is constant. In addition, a decrease in fluorescence intensity was observed under the condition where 450 µM high concentration of AHL was added.

T--TokyoTech--time-fluorescence_per_OD_char_1019_v2.png Figure: Fluorescence per OD660 of BBa K575029

The following graph shows the change in OD 660. Since we could confirm the consistent increase for 4 hours as the graph shows, we stopped the measurement at that point.

T--TokyoTech--time-OD_char_1019_v1.png Figure: OD660 of BBa K575029

[edit]
Categories
Parameters
None