Difference between revisions of "Part:BBa K575029"
(→Characterization TokyoTech 2019) |
|||
| (10 intermediate revisions by one other user not shown) | |||
| Line 22: | Line 22: | ||
Overview: | 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 <i>E.coli</i>. 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. | 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 <i>E.coli</i>. 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： | Experimental Procedure： | ||
| + | |||
・Amplify the part (BBa K575029) from iGEM 2019 Distribution Kit | ・Amplify the part (BBa K575029) from iGEM 2019 Distribution Kit | ||
| + | |||
・Transform into bacteria (DH5α) | ・Transform into bacteria (DH5α) | ||
| + | |||
・Plate the bacteria in Chloramphenicol-resistant plate | ・Plate the bacteria in Chloramphenicol-resistant plate | ||
| + | |||
| + | |||
Data Analysis： | Data Analysis： | ||
| + | |||
When successful colonies start growing, data collection can proceed. | When successful colonies start growing, data collection can proceed. | ||
| + | |||
１．Select a colony and inoculate it into 5mL of LB media with Chloramphenicol. | 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. | 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. | 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： | Result： | ||
| − | |||
| − | Note: The fluorescence intensity did not increase under the addition of 225µM | + | 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. | ||
| + | |||
| + | |||
| + | [[File:T--TokyoTech--time-fluorescence_per_OD_char_1021.png|550px|<b>Fig. 1.</b> Fluorescence per OD660 of BBa K575029]] | ||
| + | |||
| + | 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. | 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. | ||
| − | + | ||
| + | [[File:T--TokyoTech--time-OD_char_1019_v1.png|550px|<b>Fig. 2.</b>OD660 of BBa K575029]] | ||
| + | |||
Figure: OD660 of BBa K575029 | Figure: OD660 of BBa K575029 | ||
Latest revision as of 01:51, 22 October 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 58
Illegal NheI site found at 918
Illegal NheI site found at 941 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1294
Illegal AgeI site found at 1491 - 1000INCOMPATIBLE 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