Part:BBa_K4844007:Design
Low phosphate phytosensor with low noise amplifier gene circuit
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1331
Illegal XbaI site found at 277
Illegal XbaI site found at 398
Illegal PstI site found at 3145
Illegal PstI site found at 3732 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1331
Illegal NheI site found at 749
Illegal NheI site found at 3506
Illegal NheI site found at 3848
Illegal NheI site found at 6885
Illegal PstI site found at 3145
Illegal PstI site found at 3732 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1331
Illegal BglII site found at 5547
Illegal BglII site found at 8903
Illegal BamHI site found at 5313 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1331
Illegal XbaI site found at 277
Illegal XbaI site found at 398
Illegal PstI site found at 3145
Illegal PstI site found at 3732 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1331
Illegal XbaI site found at 277
Illegal XbaI site found at 398
Illegal PstI site found at 3145
Illegal PstI site found at 3732
Illegal NgoMIV site found at 3502 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3485
Illegal SapI.rc site found at 3622
Design Notes
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
Source
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.