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.
Select a colony and inoculate it into 5mL of LB media with Chloramphenicol.
Add a certain amount of PAI1 (AHL, 3-oxo-C12-HSL) so that each sample reaches the specified final concentration ranging from 0 ”M to 450 ”M.
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.
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 PAI1 (AHL) concentration. In this assay for BBa K575029, the threshold value for the AHL addition amount was between 56 ”M and 112 ”M.
Note: The fluorescence intensity did not increase under the addition of 225”M PAI1. From this, there is a threshold for the induction of PAI1 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 ”M high concentration of AHL was added.
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.
