Difference between revisions of "Part:BBa K3245007"
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<h1>BBa_K3245007:</h1>
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<h1>Fudan 2019’s characterization and improvement:</h1>
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<p>BBa_K3245007（J23106-B0034-C0040-B0015-R0040-B0034-K3245008-B0014-B0015） is one of a series of composite parts designed for characterizing R0040.</p>
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<h2>Usage and biology:</h2>
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<h2>Background:</h2>
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<p>This part is one of a collection of parts designed for characterizing ptetR (R0040). This part constantly expresses tetR(C0040) at middle-high level strength to inhibit the downstream ptetR from expressing sfGFP. When induced by aTc/tet, this part shows a leaping induction curve. To use this part, simply clone it into a middle/high copy plasmid vector, transfer into E.coli K-12, incubate overnight, and induce with aTc after proper dilution. </p>
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<p>In our project, we hope that this promoter can trigger the regulation of the downstream tetR circuit properly without leakage and subsequent deactivation. Since we use the production of its upstream regulating genes luxI and luxR as its signal in our final circuit, our measurement continued this design and measure the curve basing on OD value. Data collected in unit of the concentration of bacteria will directly reflect if the system works properly. </p>
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<h2>Experiment design and measurement:</h2>
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<p>To test this part’s function, we transferred DH10B bacteria with plasmids carrying the promoter with another LuxI-LuxR ( BBa_K3245002 ) plasmid at the same time. LuxI-LuxR is a upstream regulation box which produces signal that can activate luxpR in QS system. And we added GFP after luxpR as the reporter of its expression level. By measuring fluorescence/OD600, the expressing level of luxpR can be known and the data are comparable with our other works.</p>
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<h2>Result:</h2>
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[[File:T--Fudan--PartFinalDay1.png|700px]]
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[[File:T--Fudan--PartFinalDay2.png|700px]]
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<p>Fig.1 This figure shows luxpR-HS expression level induced by luxR-luxI box. The MEFL/particle changes as the OD goes up after dilution.</p>
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<p>At first the parental fluorescence and dilution effect keep fluorescence/OD600 at an extremely high level. Then the concentration of signal molecules remains low in the fresh culture while the bacteria are reproducing. In the next few hours the fluorescence/OD600 keeps going down. After the newly cultured bacteria grow to a higher concentration, luxpR-HS is activated and fluorescence/OD600 rises again as Fig.1 shows. Expression properties of luxpR-HS is predictable according to the fitting analysis results.</p>
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<h2>Improvement:</h2>
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<p>We substituted the -35 to -10 region of the original promoter, luxpR-HS with the one from constitutive promoter family members (BBa_J23100 :https://parts.igem.org/Part:BBa_J23100), according to the knowledge of lux box[1] and the method of constructing hybrid promoters[2]. Finally we picked luxpR-HS100 as our improved part and deployed it in our circuit.</p>
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<h2>Design:</h2>
 
<h2>Design:</h2>
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<p>Since our project involves tetR-ptetR double plasmid expression system (Fig.1), it is essential for us to characterize ptetR by measuring the level of tetR expression required for total inhibition. To achieve this, we designed 3 different tetR-ptetR expression systems (BBa_K3245007, BBa_K3245012, BBa_K3245011), among which K3245007 expresses the highest amount of tetR (J23106-B0034). </p>
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[[File:T--Fudan--PartT3.jpg|700px]]
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<p>In order not to let the promoter of antibiotics and J23106 affect the process of characterization, we inserted 2 B0015s into our expression system, one at the upstream of R0040, and the other at the downstream of sfGFP. </p>
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<p>Fig.2 Hybrid promoter diagram. As the figure shows, we substituted the -35 to -10 region of the original promoter luxpR with the one of J23100, a strong constitutive promoter. This region is rarely concerned as it’s rather conservative in promoters with the same function, but it has crucial structural effect on σ factor binding and other events in transcription regulation. Fortunately the change proved to be effective on adjusting the behavior of the regulatory promoter.</p>
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[[File:T--Fudan--Part1.png|700px]]
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<h2>Result:</h2>
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[[File:T--Fudan--PartT4.jpg|700px]]
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<p>Fig. 3 Microplate reader data of quorum sensing promoters ( time as X-axis ). It shows the overall status of the promoters. Both luxpR-HS100 and luxpR-HS show low leakage at the low OD600 period, at the platform HS100 shows higher expression level than HS.</p>
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[[File:T--Fudan--PartT5.jpg|700px]]
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<p>Fig. 4 Microplate reader data of quorum sensing promoters ( OD600 as X-axis ). The downward curve before OD600 <0.1 is due to the parental fluorescence and the dilution effect after proliferation. It is shown that luxpR-HS100 has a higher expression level and keeps low leakage similar to luxpR-HS.</p>
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<h2>Characterization:</h2>
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<h2>Protocol:</h2>
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<p>In order to characterize R0040, we cloned K3245007 into p15A, a vector with middle copy number (15-20). We then transferred p15A-K3245007 into E.coli DH10B and incubated it overnight. The culture was diluted with LB to 1/500 before induction (see team Fudan_protocol for more detail). Due to lack of access to aTc, we use tetracycline (tet) to induce R0040. Our results are shown as follows: </p>
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<p>We cultured the bacteria mentioned above overnight. As it reached a high concentration, luxpR-HS became activated. Then we diluted the culture to 1/5000 and measured its fluorescence and OD600 every 30 minutes.</p>
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[[File:T--Fudan--PartFinalDay1.jpg|600px]]
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<p>1. Inoculate the selected single colony with 4 kinds of luxpR promoters above into 3 ml LB medium with 50 mg/ml kanamycin and 100 mg/ml ampicillin at 37℃.</p><p>2. Dilute the overnight culture to 1/5000 in 15ml fresh LB medium with 50 mg/ml kanamycin and 100mg/ml ampicillin. Each group with two parallel copies.</p>
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<p>Fig.1 induction curve of K3245007 (7 hours). Data are collected and analyzed according to iGEM standard data analysis form after 7 hours of induction. </p>
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<p>3. Incubate all culture above at 37℃.</p>
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[[File:T--Fudan--PartFinalDay2.jpg|700px]]
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<p>4. Pipette 200 μl each as sample every 30 minutes and measure their fluorescence and OD600.</p>
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<p>Fig.2 figure showing different induction curves of K3245007 under different tet concentration with the lapse of time. Data are collected and analyzed according to iGEM standard data analysis form after 7 hours of induction.</p>
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<h2>Reference:</h2>
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<p>[1] Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.</p>
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<p>[2] de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983;80(1):21–25. doi:10.1073/pnas.80.1.21</p>
  
  

Revision as of 20:00, 21 October 2019

Fudan 2019’s characterization and improvement:

Background:

In our project, we hope that this promoter can trigger the regulation of the downstream tetR circuit properly without leakage and subsequent deactivation. Since we use the production of its upstream regulating genes luxI and luxR as its signal in our final circuit, our measurement continued this design and measure the curve basing on OD value. Data collected in unit of the concentration of bacteria will directly reflect if the system works properly.

Experiment design and measurement:

To test this part’s function, we transferred DH10B bacteria with plasmids carrying the promoter with another LuxI-LuxR ( BBa_K3245002 ) plasmid at the same time. LuxI-LuxR is a upstream regulation box which produces signal that can activate luxpR in QS system. And we added GFP after luxpR as the reporter of its expression level. By measuring fluorescence/OD600, the expressing level of luxpR can be known and the data are comparable with our other works.


Result:

700px 700px

Fig.1 This figure shows luxpR-HS expression level induced by luxR-luxI box. The MEFL/particle changes as the OD goes up after dilution.

At first the parental fluorescence and dilution effect keep fluorescence/OD600 at an extremely high level. Then the concentration of signal molecules remains low in the fresh culture while the bacteria are reproducing. In the next few hours the fluorescence/OD600 keeps going down. After the newly cultured bacteria grow to a higher concentration, luxpR-HS is activated and fluorescence/OD600 rises again as Fig.1 shows. Expression properties of luxpR-HS is predictable according to the fitting analysis results.

Improvement:

We substituted the -35 to -10 region of the original promoter, luxpR-HS with the one from constitutive promoter family members (BBa_J23100 :https://parts.igem.org/Part:BBa_J23100), according to the knowledge of lux box[1] and the method of constructing hybrid promoters[2]. Finally we picked luxpR-HS100 as our improved part and deployed it in our circuit.

Design:

700px

Fig.2 Hybrid promoter diagram. As the figure shows, we substituted the -35 to -10 region of the original promoter luxpR with the one of J23100, a strong constitutive promoter. This region is rarely concerned as it’s rather conservative in promoters with the same function, but it has crucial structural effect on σ factor binding and other events in transcription regulation. Fortunately the change proved to be effective on adjusting the behavior of the regulatory promoter.


Result:

700px

Fig. 3 Microplate reader data of quorum sensing promoters ( time as X-axis ). It shows the overall status of the promoters. Both luxpR-HS100 and luxpR-HS show low leakage at the low OD600 period, at the platform HS100 shows higher expression level than HS.

700px

Fig. 4 Microplate reader data of quorum sensing promoters ( OD600 as X-axis ). The downward curve before OD600 <0.1 is due to the parental fluorescence and the dilution effect after proliferation. It is shown that luxpR-HS100 has a higher expression level and keeps low leakage similar to luxpR-HS.


Protocol:

We cultured the bacteria mentioned above overnight. As it reached a high concentration, luxpR-HS became activated. Then we diluted the culture to 1/5000 and measured its fluorescence and OD600 every 30 minutes.

1. Inoculate the selected single colony with 4 kinds of luxpR promoters above into 3 ml LB medium with 50 mg/ml kanamycin and 100 mg/ml ampicillin at 37℃.

2. Dilute the overnight culture to 1/5000 in 15ml fresh LB medium with 50 mg/ml kanamycin and 100mg/ml ampicillin. Each group with two parallel copies.

3. Incubate all culture above at 37℃.

4. Pipette 200 μl each as sample every 30 minutes and measure their fluorescence and OD600.

Reference:

[1] Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.

[2] de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983;80(1):21–25. doi:10.1073/pnas.80.1.21


Part constructed to characterize part R0040.

As we use tetR-ptetR system in our project, it is essential for us to measure the minimum amount of tetR needed to stop ptetR. Considering that ptetR is a rather strict regulatory promoter, we constructed 4 tetR-ptetR-GFP expression systems,each with different strength of tetR. Then we measured both their initial GFP strength as well as their induction curve.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]