Difference between revisions of "Part:BBa K3887008"
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This is an optimized part of [[Part:BBa_K2406020|BBa_K2406020]] .
 
This is an optimized part of [[Part:BBa_K2406020|BBa_K2406020]] .
  
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=Decriptioon=
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=Description=
  
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T7 promoter is commonly used in iGEM projects, [Part:BBa_K2406020|BBa_K2406020]] designed by iGEM17_Edinburgh_UG was an inducile T7-LacO promoter for gene expression. In our project, we also used T7-LacO promoter to control the expression of halogenase. However, we found high basic leakage of this promoter, which affected the results of our experiments, so we improved this promoter in our project.
+
T7 promoter is commonly used in iGEM projects, [[Part:BBa_K2406020|BBa_K2406020]] designed by iGEM17_Edinburgh_UG was an inducile T7-LacO promoter for gene expression. In our project, we also used T7-LacO promoter to control the expression of halogenase. However, we found high basic leakage of this promoter, which affected the results of our experiments, so we improved this promoter in our project.
 
It is recognized that an excellent promoter should possess exceptional switch ratio: it should have lower background leakage in the "off" state and have obvious expression in the "on" state. In order to reduce the leakage and to ensure the repressor can bind better to the LacO site, we added another LacO site in the T7-LacO promoter as the figure below shows:
 
It is recognized that an excellent promoter should possess exceptional switch ratio: it should have lower background leakage in the "off" state and have obvious expression in the "on" state. In order to reduce the leakage and to ensure the repressor can bind better to the LacO site, we added another LacO site in the T7-LacO promoter as the figure below shows:
 
[[File:T--BJU_China--Fig.1 mprovement.png|600px|thumb|center]]
 
[[File:T--BJU_China--Fig.1 mprovement.png|600px|thumb|center]]
 
<p  class="figure-description"><b><center>Fig.1 Improved promoter and original promoter</center></b></p>
 
<p  class="figure-description"><b><center>Fig.1 Improved promoter and original promoter</center></b></p>
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 +
=Gene Circuit and Test Method=
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In the experiment, LacI was designed as a repressor and IPTG as an inducer to control the state of T7 promoter. The gene circuit diagram is shown in Fig. 2. At the same time, we use sfGFP as reporter gene, so that the fluorescence value can be used to quantitatified gene expression level.
 +
[[File:T--BJU_China--Fig.2 improvement gene circuit.png|600px|thumb|center]]
 +
<p  class="figure-description"><b><center>Fig.2 Gene circuit for promoter test</center></b></p>
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Single colonies of two bacteria (control, improved) were inoculated into LB medium, and cultivated vernight at 37° as seed culture. The seed culture was inoculated into M9 medium as 1:100, and IPTG were added at 0h respectively. The concentration of IPTG was 0, 10, 20, 50, 100, 200, 500, 1000 μM, and three parallel samples for each concentration. 200 μl of each sample were taken to measure the fluorescence after 6 hours of induction.
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 +
=Results and Discussion=
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From the results shown in Fig.3，we can see that the improved T7-LacO promoter is much better than the control group in terms of lower basic leakage or higher expression status. At the zero point，that is without inducer, the leakage of improved  promoter is more than ten times lower than the control group. While when enough inducer exist, that is, when the gene circuit is fully turned on, the maximum expression of the optimized promoter is not suppressed, and even slightly higher than the control group.
 +
Furthermore, when comparing the switch ratios of the two inducible promoters, the ratio of the control group was only about 12, while the switch ratio of improved promoter could reach 147, which also shows that our modification of this part is effective.
 +
[[File:T--BJU_China--Fig.3 mprovement results.png|600px|thumb|center]]
 +
<p  class="figure-description"><b><center>Fig.3 Performance of improved promoter and original promoter</center></b></p>
 +
In the above experiments and results, we optimized the characterization performance of the T7-LacO promoter by adding an extra LacO site. This may be because in the lactose operon model, the LacI repressor is a tetramer that binds to the LacO site to control the state of inducible promoter. The function of LacO involves various factors such as DNA bending and interaction between protein and DNA. The specific influence mechanism of the amount of lacO on the promoter needs to be further studied.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 08:12, 13 October 2021


LacO-scar-T7-LacO Promoter

This is an optimized part of BBa_K2406020 .

Description

T7 promoter is commonly used in iGEM projects, BBa_K2406020 designed by iGEM17_Edinburgh_UG was an inducile T7-LacO promoter for gene expression. In our project, we also used T7-LacO promoter to control the expression of halogenase. However, we found high basic leakage of this promoter, which affected the results of our experiments, so we improved this promoter in our project. It is recognized that an excellent promoter should possess exceptional switch ratio: it should have lower background leakage in the "off" state and have obvious expression in the "on" state. In order to reduce the leakage and to ensure the repressor can bind better to the LacO site, we added another LacO site in the T7-LacO promoter as the figure below shows:

T--BJU China--Fig.1 mprovement.png

Fig.1 Improved promoter and original promoter

Gene Circuit and Test Method

In the experiment, LacI was designed as a repressor and IPTG as an inducer to control the state of T7 promoter. The gene circuit diagram is shown in Fig. 2. At the same time, we use sfGFP as reporter gene, so that the fluorescence value can be used to quantitatified gene expression level.

T--BJU China--Fig.2 improvement gene circuit.png

Fig.2 Gene circuit for promoter test

Single colonies of two bacteria (control, improved) were inoculated into LB medium, and cultivated vernight at 37° as seed culture. The seed culture was inoculated into M9 medium as 1:100, and IPTG were added at 0h respectively. The concentration of IPTG was 0, 10, 20, 50, 100, 200, 500, 1000 μM, and three parallel samples for each concentration. 200 μl of each sample were taken to measure the fluorescence after 6 hours of induction.

Results and Discussion

From the results shown in Fig.3，we can see that the improved T7-LacO promoter is much better than the control group in terms of lower basic leakage or higher expression status. At the zero point，that is without inducer, the leakage of improved promoter is more than ten times lower than the control group. While when enough inducer exist, that is, when the gene circuit is fully turned on, the maximum expression of the optimized promoter is not suppressed, and even slightly higher than the control group. Furthermore, when comparing the switch ratios of the two inducible promoters, the ratio of the control group was only about 12, while the switch ratio of improved promoter could reach 147, which also shows that our modification of this part is effective.

T--BJU China--Fig.3 mprovement results.png

Fig.3 Performance of improved promoter and original promoter

In the above experiments and results, we optimized the characterization performance of the T7-LacO promoter by adding an extra LacO site. This may be because in the lactose operon model, the LacI repressor is a tetramer that binds to the LacO site to control the state of inducible promoter. The function of LacO involves various factors such as DNA bending and interaction between protein and DNA. The specific influence mechanism of the amount of lacO on the promoter needs to be further studied.

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]