Part:BBa_K575029

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 PAI1 (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 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 µM 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.

Figure: OD660 of BBa K575029