Difference between revisions of "Part:BBa K4844007"

 
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<partinfo>BBa_K4844005 short</partinfo>
 
<partinfo>BBa_K4844005 short</partinfo>
  
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Usage:
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=Usage=
 
Plants usually have severe responses under the pressure of low phosphate. Some genes will be turned on to  allow plants to better adapt to this situation. Therefore, in order to build a low-cost, real-time soil phosphate sensor, we first found a low phosphate response promoter in Maize which was reported by Jianrong Bai in 2018. Based on Bai's result, a 1502bp length promoter P1502-ZmPHR1 (BBa_K4844002, a new part submitted by our team in 2023) shows the strongest promoting strength under low phosphate pressure. Therefore, we decided to choose it as our low phosphate detection part. The sensor vector will be transformed into plants later with our "TTTT" system. (More about TTTT can be found at https://2023.igem.wiki/sz-shd/plant )
 
Plants usually have severe responses under the pressure of low phosphate. Some genes will be turned on to  allow plants to better adapt to this situation. Therefore, in order to build a low-cost, real-time soil phosphate sensor, we first found a low phosphate response promoter in Maize which was reported by Jianrong Bai in 2018. Based on Bai's result, a 1502bp length promoter P1502-ZmPHR1 (BBa_K4844002, a new part submitted by our team in 2023) shows the strongest promoting strength under low phosphate pressure. Therefore, we decided to choose it as our low phosphate detection part. The sensor vector will be transformed into plants later with our "TTTT" system. (More about TTTT can be found at https://2023.igem.wiki/sz-shd/plant )
  
 
When the plant was under low phosphate stress. The P1502-ZmPHR1 promoter will up-regulate its' expression, the signal then enters the amplifier system, which eventually yields a large quantity of super bright eyGFP_UV (BBa_K4844000, new basic part submitted by our team in 2023) protein that can be seen under UV light with the naked eye.
 
When the plant was under low phosphate stress. The P1502-ZmPHR1 promoter will up-regulate its' expression, the signal then enters the amplifier system, which eventually yields a large quantity of super bright eyGFP_UV (BBa_K4844000, new basic part submitted by our team in 2023) protein that can be seen under UV light with the naked eye.
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 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-1.png"></html>
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 +
=Biology: design=
 +
 +
1. Design of Low phosphate sensor (Basic)
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-2.png"></html>
 +
 +
By combining the P1502-ZmPHR1 promoter with the eyGFP report system we already introduced. Also, a normally open GUS gene as the internal reference for downstream experiments (to calibrate the result). We get the basic version of the low phosphate sensor.
 +
 +
2. Design of Low phosphate sensor (with Low noise amplifier)
 +
Although the P1502-ZmPHR1 promoter in the basic version up-regulates the expression when the plant is under low phosphate pressure. However, we found that the leaking expression (background expression without low phosphate pressure) of the original promoter was high and the promoting strength wasn't as high as we expected. Therefore, we decided to design a signal amplification system.
 +
 +
Therefore, we designed an artificial transcription factor to regulate artificial promoters, aiming to enhance the low-phosphate response in plants. In this artificial transcription factor, we split the DNA binding domain (DBD) and the transcription activation domain into two separate proteins, placing them downstream of two relatively weaker promoters. We then used the N-terminal (N) and C-terminal (C) protein-protein interaction domains (PID) to guide the recombination and activation of the split transcription factors with their respective artificial promoters.
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-3.png"></html>
 +
 +
The complete low-phosphate amplification circuit design is shown in the figure below, where LacI-PDZ and TP-VP16 are placed under the control of the pZmPHR1 and pZmSO promoters. When both promoters are simultaneously activated, LacI-PDZ and TP-VP16 proteins bind to the lacO sequence on the artificial promoter 35SE-lacO-mini35p, activating the downstream fluorescent protein gene transcription. Additionally, when LacI-PDZ is expressed alone, it's binding to DNA can block certain promoter leaky expressions, making this system more controllable.
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-4.png"></html>
 +
 +
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-5.png"></html>
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 +
=Characterization:=
 +
Hence, we validate our design through different ways. ( Detailed protocols can be found on the supplementary material page https://2023.igem.wiki/sz-shd/experiments ).
 +
1. Successful in vitro validation of LacI-LacO binding with electrophoretic mobility shift assay (EMSA)
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 +
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-6.png"></html>
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Our result proved that the LacI protein can bind to the LacO DNA sequence as we designed.
 +
 +
2.  qPCR results indicate the function of our gene circuit at the transcription level
 +
To verify the function of the amplification gene circuit, we decided to use qPCR- a semi-quantitative strategy to measure transcription efficiency.
 +
 +
 +
 +
 +
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-7.png"></html>
 +
 +
The qPCR result indicates that our low noise amplifier part can not only increase the expression strength but also reduce leaky expression.
 +
 +
3. Live visualization of eyGFP(UV) under UV flashlight
 +
To test the real-world application of our product, we use a UV flashlight to visualize the eyGFP(UV) of the carbon dots transformed tobacco leaves after 5 days of low phosphate treatment.
 +
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-8.png"></html>
 +
 +
 +
 +
 +
Therefore, our phytosensor design showed engineering success and has the potential to turn into a product and application in agricultural production. We also validate the potential logic gates which can be used in plants.
 +
 +
For detailed design about this part, visit: https://2023.igem.wiki/sz-shd/engineering#construction
 +
 +
<html><img src="https://static.igem.wiki/teams/4844/wiki/parts/bba-k4844005-9.png"></html>
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All plasmid files can be downloaded at: https://2023.igem.wiki/sz-shd/experiments
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 14:19, 12 October 2023


Low phosphate phytosensor with low noise amplifier gene circuit

Usage

Plants usually have severe responses under the pressure of low phosphate. Some genes will be turned on to allow plants to better adapt to this situation. Therefore, in order to build a low-cost, real-time soil phosphate sensor, we first found a low phosphate response promoter in Maize which was reported by Jianrong Bai in 2018. Based on Bai's result, a 1502bp length promoter P1502-ZmPHR1 (BBa_K4844002, a new part submitted by our team in 2023) shows the strongest promoting strength under low phosphate pressure. Therefore, we decided to choose it as our low phosphate detection part. The sensor vector will be transformed into plants later with our "TTTT" system. (More about TTTT can be found at https://2023.igem.wiki/sz-shd/plant )

When the plant was under low phosphate stress. The P1502-ZmPHR1 promoter will up-regulate its' expression, the signal then enters the amplifier system, which eventually yields a large quantity of super bright eyGFP_UV (BBa_K4844000, new basic part submitted by our team in 2023) protein that can be seen under UV light with the naked eye.

Biology: design

1. Design of Low phosphate sensor (Basic)

By combining the P1502-ZmPHR1 promoter with the eyGFP report system we already introduced. Also, a normally open GUS gene as the internal reference for downstream experiments (to calibrate the result). We get the basic version of the low phosphate sensor.

2. Design of Low phosphate sensor (with Low noise amplifier) Although the P1502-ZmPHR1 promoter in the basic version up-regulates the expression when the plant is under low phosphate pressure. However, we found that the leaking expression (background expression without low phosphate pressure) of the original promoter was high and the promoting strength wasn't as high as we expected. Therefore, we decided to design a signal amplification system.

Therefore, we designed an artificial transcription factor to regulate artificial promoters, aiming to enhance the low-phosphate response in plants. In this artificial transcription factor, we split the DNA binding domain (DBD) and the transcription activation domain into two separate proteins, placing them downstream of two relatively weaker promoters. We then used the N-terminal (N) and C-terminal (C) protein-protein interaction domains (PID) to guide the recombination and activation of the split transcription factors with their respective artificial promoters.

The complete low-phosphate amplification circuit design is shown in the figure below, where LacI-PDZ and TP-VP16 are placed under the control of the pZmPHR1 and pZmSO promoters. When both promoters are simultaneously activated, LacI-PDZ and TP-VP16 proteins bind to the lacO sequence on the artificial promoter 35SE-lacO-mini35p, activating the downstream fluorescent protein gene transcription. Additionally, when LacI-PDZ is expressed alone, it's binding to DNA can block certain promoter leaky expressions, making this system more controllable.


Characterization:

Hence, we validate our design through different ways. ( Detailed protocols can be found on the supplementary material page https://2023.igem.wiki/sz-shd/experiments ). 1. Successful in vitro validation of LacI-LacO binding with electrophoretic mobility shift assay (EMSA)



Our result proved that the LacI protein can bind to the LacO DNA sequence as we designed.

2. qPCR results indicate the function of our gene circuit at the transcription level To verify the function of the amplification gene circuit, we decided to use qPCR- a semi-quantitative strategy to measure transcription efficiency.




The qPCR result indicates that our low noise amplifier part can not only increase the expression strength but also reduce leaky expression.

3. Live visualization of eyGFP(UV) under UV flashlight To test the real-world application of our product, we use a UV flashlight to visualize the eyGFP(UV) of the carbon dots transformed tobacco leaves after 5 days of low phosphate treatment.




Therefore, our phytosensor design showed engineering success and has the potential to turn into a product and application in agricultural production. We also validate the potential logic gates which can be used in plants.

For detailed design about this part, visit: https://2023.igem.wiki/sz-shd/engineering#construction

All plasmid files can be downloaded at: https://2023.igem.wiki/sz-shd/experiments


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 709
    Illegal NheI site found at 2259
    Illegal NheI site found at 5016
    Illegal NheI site found at 5358
    Illegal NheI site found at 8395
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 7057
    Illegal BglII site found at 10413
    Illegal BamHI site found at 6823
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5012
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4995
    Illegal SapI site found at 11505
    Illegal SapI.rc site found at 5132