Difference between revisions of "Part:BBa K4043004"

Latest revision as of 09:09, 9 October 2022

A weak promoter

The transcription of the PsacB promoter is not strictly regulated by sucrose, and can be transcribed without an inducer, but the intensity is 100 times lower than that of sucrose induction.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

User Reviews

Contribution

Group: ZJU-China 2022
Author: Zhijian Yan

Summary: In 2022, ZJU-China characterized this part and used this part in their project to design a kill switch regulated by the concentration of sucrose(BBa_K4202045). The characterization result indicated the promoter activity is too low to permit reliable characterization result when using GFP as reporter. The kill switch containing this promoter has been proved to work very well in Bacillus subtilis WB600 strain.

Characterization

To measure activity of this part, we created the measurement part BBa_K4202029 and then constructed it to the vector pHY300PLK and transformed it to Bacillus subtilis WB600 strain. The bacteria containing the measurement plasmid were than cultured in the TB media and induced by different concentrations of sucrose and sucralose(a sucrose analogue).The fluorescence intensity of engineered bacteria were measured using Flow Cytometer

Fig 1-1 The output of fluorescence intensity using different types and concentrations of inducers. SUC:sucrose, SUL:sucralose

The result showed that when sucrose and sucralose were used as inducers, the PsacB will saturate when the inducer concentration reaches 0.5%. Higher concentration of inducers may create excessive osmotic pressure and apply excessive metabolic burden for the bacteria, which can lead to the increase of activity. What's more, using sucrose as an inducer can lead up to ~5 fold difference in activity, much higher than the ~2-fold difference in activity produced by the sucralose.

However, it is worth noting that since the fluorescence intensity produced by the bacteria in this experiment is really close to the detection limit of flow cytometer, the results can only indicate the trend of the correlation between the inducer concentration and promoter activity, and cannot be used as an absolute quantitative basis. We also construct two other measurement parts ((BBa_K4202031) and (BBa_K4202032)using Luciferase and NanoLuc as reporter, but we did not complete all the experiments due to time constraints. The newest characterization result will be uploaded after the experiment is finished.

Improvement

We construct a set of engineered promoters through replacing the constitutive promoter with a stronger constitutive promoter Pveg and multipling the leader RNA of PsacB for different times. Here is our engineered promoters: BBa_K4202025, BBa_K4202026, BBa_K4202027, BBa_K4202028.

Application

For more information about how we use this part in our kill switch, please turn to the experience page or our part BBa_K4202045.

@@ Line 25: / Line 25: @@
 <br>
 <b>Characterization</b>
-<p>To measure activity of this part, we created the measurement part <b><partinfo>BBa_K4202029</partinfo></b> and then constructed it to the  vector pHY300PLK and transformed it to <i>Bacillus subtilis</i> WB600 strain. The bacteria containing the measurement plasmid were than cultured in the TB media and induced by different concentrations of sucrose and sucralose(a sucrose analogue).</p>
+<p>To measure activity of this part, we created the measurement part <b><partinfo>BBa_K4202029</partinfo></b> and then constructed it to the  vector pHY300PLK and transformed it to <i>Bacillus subtilis</i> WB600 strain. The bacteria containing the measurement plasmid were than cultured in the TB media and induced by different concentrations of sucrose and sucralose(a sucrose analogue).The fluorescence intensity of engineered bacteria were measured using Flow Cytometer</p>
-[[File:ZJU-China2022-PsacB measurement result.png]]
+<div align="center">[[File:ZJU-China2022-PsacB-measurement-result.png|600px|]]</div>
+<br>
+<div align="center"><b>Fig 1-1</b> The output of fluorescence intensity using different types and concentrations of inducers.
+SUC:sucrose, SUL:sucralose</div>
+<br>
+<p>The result showed that when sucrose and sucralose were used as inducers, the PsacB will saturate when the inducer concentration reaches 0.5%. Higher concentration of inducers may create excessive osmotic pressure and apply excessive metabolic burden for the bacteria, which can lead to the increase of activity. What's more, using sucrose as an inducer can lead up to ~5 fold difference in activity, much higher than the ~2-fold difference in activity produced by the sucralose.</p>
+<p>However, it is worth noting that since the fluorescence intensity produced by the bacteria in this experiment is really close to the detection limit of flow cytometer, the results can only indicate the trend of the correlation between the inducer concentration and promoter activity, and cannot be used as an absolute quantitative basis. We also construct two other measurement parts ((<b><partinfo>BBa_K4202031</partinfo></b>) and (<b><partinfo>BBa_K4202032</partinfo></b>)using Luciferase and NanoLuc as reporter, but we did not complete all the experiments due to time constraints. The newest characterization result will be uploaded after the experiment is finished.</p>
+<br>
+<b>Improvement </b>
+<p>We construct a set of engineered promoters through replacing the constitutive promoter with a stronger constitutive promoter Pveg and multipling the leader RNA of PsacB for different times. Here is our engineered promoters: <b><partinfo>BBa_K4202025</partinfo></b>,
+<b><partinfo>BBa_K4202026</partinfo></b>, <b><partinfo>BBa_K4202027</partinfo></b>, <b><partinfo>BBa_K4202028</partinfo></b>.</p>
+<b>Application</b>
+<p>For more information about how we use this part in our kill switch, please turn to the experience page or our part <b><partinfo>BBa_K4202045</partinfo></b>. </p>