Difference between revisions of "Part:BBa K5375007"

 
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<partinfo>BBa_K5375007 short</partinfo>
 
<partinfo>BBa_K5375007 short</partinfo>
  
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siHSP70-2
 
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5375007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5375007 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K5375007 parameters</partinfo>
 
<partinfo>BBa_K5375007 parameters</partinfo>
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__TOC__
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<span id="origin"></span>
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= Origin =
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Synthesized by company.
 +
 
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<span id="properties"></span>
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= Properties =
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Inhibition of Heat Shock Protein 70 expression.
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<span id="usage-and-biology"></span>
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= Usage and Biology =
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siHSP70-2 inhibits the target gene HSP70 as a small interfering RNA (siRNA). HSP70 can induce IgE-mediated hypersensitivity reactions and T-cell responses in allergic individuals (Fagotti et al., 2022). Further discussion of it as a pan-allergen can be found in part BBa_K2619011. siRNA is a key component of the RNAi process, a powerful gene silencing mechanism. Once introduced into the target cells, it is recognized and loaded into the RNA-Induced Silencing Complex (RISC). The siRNA’s antisense strand binds to the complementary target mRNA molecule, triggering the RISC complex to cleave the target mRNA. This prevents the mRNA from being translated into a functional protein (Agrawal et al., 2003). The silencing effect typically lasts around 12 days for this part.
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siHSP70-2 is particularly useful in plant cells, where it successfully inhibits the expression of the pan-allergen HSP70, reducing allergic symptoms related to *Populus tomentosa* pollen allergy.
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 +
<span id="cultivation-purification"></span>
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= Cultivation and Purification =
 +
 
 +
siHSP70-2 is synthesized through oligonucleotides with a nucleic acid synthesizer. The following sequences represent the sense and antisense strands of the siRNA:
 +
 
 +
- Oligo Sequence for siHSP70-2-SS: CCUACGGUCUUGACAAGAAGC
 +
- Oligo Sequence for siHSP70-2-AS: UUCUUGUCAAGACCGUAGGCA
 +
 
 +
These oligonucleotides are then annealed to form a double-stranded siRNA molecule. The siRNA is purified using high-performance liquid chromatography (HPLC) (Sohail et al., 2003). To enhance delivery into plant cells, carbon dots (CDs) were incorporated with Polyethyleneimine (PEI) through the microwave method, which allowed the negatively charged siRNA to bind to the CDs.
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<span id="measurement-characterization"></span>
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= Measurement and Characterization =
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<html>
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<div style="text-align:center;">
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    <img src="https://static.igem.wiki/teams/5375/bba-k5375007/1.png" width="50%" style="display:block; margin:auto;" alt="RT-qPCR results for protoplasts" >
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    <div style="text-align:center;">
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        <caption>Figure 1. RT-qPCR results for protoplasts.</caption>
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    </div>
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</div>
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</html>
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The chart demonstrates the performance of siHSP70-2 in inhibiting HSP70 expression. Results indicate that siHSP70-2 successfully repressed HSP70 expression, reducing it to less than 20% of its original level.
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375007/2.png" width="50%" style="display:block; margin:auto;" alt="RT-qPCR results for tobacco leaf injection" >
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    <div style="text-align:center;">
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        <caption>Figure 2. RT-qPCR results for tobacco leaf injection.</caption>
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    </div>
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</div>
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</html>
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RT-qPCR results for siRNA injection in tobacco leaves. While siHSP70-2 showed some success in repressing HSP70 expression after combination with CDs, it was less efficient compared to siHSP70-3.
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375007/3.png" width="50%" style="display:block; margin:auto;" alt="RT-qPCR results for osmanthus tree trunk injection" >
 +
    <div style="text-align:center;">
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        <caption>Figure 3. RT-qPCR results for osmanthus tree trunk injection.</caption>
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    </div>
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</div>
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</html>
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 +
RT-qPCR results for siRNA delivery through trunk injection in osmanthus trees. Some success was observed in inhibiting HSP70 expression with the combination of CDs, but the efficacy was not as high as that of CDs@siHSP70-3.
 +
 
 +
Further trials and improvements are needed for siHSP70-2 to ensure optimal quality and efficiency in target gene repression.
 +
 
 +
<span id="reference"></span>
 +
= Reference =
 +
 
 +
Agrawal N. N., Dasaradhi P. V., Mohmmed A., Malhotra P., Bhatnagar R. K., & Mukherjee S. K. (2003). RNA Interference: Biology, Mechanism, and Applications. *Microbiology and Molecular Biology Reviews*, 67(4), 657-685. [https://doi.org/10.1128/MMBR.67.4.657-685.2003](https://doi.org/10.1128/MMBR.67.4.657-685.2003)
 +
 
 +
Fagotti A., Lucentini L., Simoncelli F., La Porta G., Brustenga L., Bizzarri I., Trio S., Isidori C., Di Rosa I., & Di Cara G. (2022). HSP70 upregulation in nasal mucosa of symptomatic children with allergic rhinitis and potential risk of asthma development. *Scientific Reports*, 12(1), 1-10. [https://doi.org/10.1038/s41598-022-18443-x](https://doi.org/10.1038/s41598-022-18443-x)
 +
 
 +
Sohail M., Doran G., Riedemann J., Macaulay V., & Southern E. M. (2003). A simple and cost-effective method for producing small interfering RNAs with high efficacy. *Nucleic Acids Research*, 31(7), e38.

Revision as of 11:11, 25 September 2024

siHSP70-2



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]


Origin

Synthesized by company.

Properties

Inhibition of Heat Shock Protein 70 expression.

Usage and Biology

siHSP70-2 inhibits the target gene HSP70 as a small interfering RNA (siRNA). HSP70 can induce IgE-mediated hypersensitivity reactions and T-cell responses in allergic individuals (Fagotti et al., 2022). Further discussion of it as a pan-allergen can be found in part BBa_K2619011. siRNA is a key component of the RNAi process, a powerful gene silencing mechanism. Once introduced into the target cells, it is recognized and loaded into the RNA-Induced Silencing Complex (RISC). The siRNA’s antisense strand binds to the complementary target mRNA molecule, triggering the RISC complex to cleave the target mRNA. This prevents the mRNA from being translated into a functional protein (Agrawal et al., 2003). The silencing effect typically lasts around 12 days for this part.

siHSP70-2 is particularly useful in plant cells, where it successfully inhibits the expression of the pan-allergen HSP70, reducing allergic symptoms related to *Populus tomentosa* pollen allergy.

Cultivation and Purification

siHSP70-2 is synthesized through oligonucleotides with a nucleic acid synthesizer. The following sequences represent the sense and antisense strands of the siRNA:

- Oligo Sequence for siHSP70-2-SS: CCUACGGUCUUGACAAGAAGC - Oligo Sequence for siHSP70-2-AS: UUCUUGUCAAGACCGUAGGCA

These oligonucleotides are then annealed to form a double-stranded siRNA molecule. The siRNA is purified using high-performance liquid chromatography (HPLC) (Sohail et al., 2003). To enhance delivery into plant cells, carbon dots (CDs) were incorporated with Polyethyleneimine (PEI) through the microwave method, which allowed the negatively charged siRNA to bind to the CDs.

Measurement and Characterization

RT-qPCR results for protoplasts
Figure 1. RT-qPCR results for protoplasts.

The chart demonstrates the performance of siHSP70-2 in inhibiting HSP70 expression. Results indicate that siHSP70-2 successfully repressed HSP70 expression, reducing it to less than 20% of its original level.

RT-qPCR results for tobacco leaf injection
Figure 2. RT-qPCR results for tobacco leaf injection.

RT-qPCR results for siRNA injection in tobacco leaves. While siHSP70-2 showed some success in repressing HSP70 expression after combination with CDs, it was less efficient compared to siHSP70-3.

RT-qPCR results for osmanthus tree trunk injection
Figure 3. RT-qPCR results for osmanthus tree trunk injection.

RT-qPCR results for siRNA delivery through trunk injection in osmanthus trees. Some success was observed in inhibiting HSP70 expression with the combination of CDs, but the efficacy was not as high as that of CDs@siHSP70-3.

Further trials and improvements are needed for siHSP70-2 to ensure optimal quality and efficiency in target gene repression.

Reference

Agrawal N. N., Dasaradhi P. V., Mohmmed A., Malhotra P., Bhatnagar R. K., & Mukherjee S. K. (2003). RNA Interference: Biology, Mechanism, and Applications. *Microbiology and Molecular Biology Reviews*, 67(4), 657-685. [1](https://doi.org/10.1128/MMBR.67.4.657-685.2003)

Fagotti A., Lucentini L., Simoncelli F., La Porta G., Brustenga L., Bizzarri I., Trio S., Isidori C., Di Rosa I., & Di Cara G. (2022). HSP70 upregulation in nasal mucosa of symptomatic children with allergic rhinitis and potential risk of asthma development. *Scientific Reports*, 12(1), 1-10. [2](https://doi.org/10.1038/s41598-022-18443-x)

Sohail M., Doran G., Riedemann J., Macaulay V., & Southern E. M. (2003). A simple and cost-effective method for producing small interfering RNAs with high efficacy. *Nucleic Acids Research*, 31(7), e38.