Difference between revisions of "Part:BBa K4147002"
 
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<partinfo>BBa_K4147002 short</partinfo>
 
<partinfo>BBa_K4147002 short</partinfo>
  
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This part contains the linear construct BBa_K4147001 for (CBD)2-DrsB1 which already counts with a PelB signal peptide on the N- terminal as well as a 6X-His tag at the end of the dermaseptin CDS (C- terminal) for later protein purification.  This composite construct includes a LacI regulated promoter. Additionally, it contains an RBS under the part name BBa_B0032. Finally, it has a double terminator (BBa_B0015) to ensure correct termination. As a linear 1075 bp construct, it contains the RFC10 prefix and suffix, making the part compatible with other iGEM parts.  
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This part contains the linear construct BBa_K4147001 for (CBD)2-DrsB1 which already incorporates a PelB signal peptide on the N- terminal as well as a 6X-His tag at the end of the dermaseptin CDS (C- terminal) for later protein purification.  This composite construct includes a LacI regulated promoter. Additionally, it contains an RBS under the part name BBa_B0032. Finally, it has a double terminator (BBa_B0015) to ensure correct termination. As a linear 1075 bp construct, it contains the RFC10 prefix and suffix, making the part compatible with other iGEM parts.  
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Among a wide range of AMPs, polycationic dermaseptin peptides are produced by the skin glands of the frog <i>Phyllomedusa bicolor</i> [1]. It supplies both antibacterial and antifungal protection to a broad range of plant pathogens but shows no toxic effects on plant and mammalian cells [2]. This peptide of molecular weight of 22.1 kDa contains a tandem repeat of a chitin binding domain (CBD) as an improvement of dermaseptin-b1 gene (AKA MsrA2)(BBa_K2577001) which binds to chitin fungal cell wall, thereby increasing the lytic activity of the catalytic dom
 
  
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===Usage and Biology===
 
===Usage and Biology===
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<p align="justify">
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Among a wide range of AMPs, polycationic dermaseptin peptides are produced by the skin glands of the frog <i>Phyllomedusa bicolor</i>. Although most dermaseptins are known to disrupt pathogens' lipid membranes, which leads to the development of membrane diseases, some may breach the membrane and interfere with critical cell components like DNA [1]. This system makes this AMPs the alternative antibiotics to boost plant innate immune system. It supplies both antibacterial and antifungal protection to a broad range of plant pathogens but shows no toxic effects on plant and mammalian cells [2]. This peptide of molecular weight of 22.1 kDa contains a tandem repeat of a chitin binding domain (CBD) as an improvement of dermaseptin-b1 gene (AKA MsrA2)(BBa_K2577001) which binds to chitin fungal cell wall, thereby increasing the lytic activity of the catalytic domain.</p>
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<p align="justify">
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This fusion of DrsB1 to CBD not only protect DrsB1 from host degradation but its affinity for fungal cell wall chitin remains the same. It has been shown showed that this recombinant peptide possessed strong antifungal activity <i>in vitro</i>, inhibiting the growth of mycelia as well as the germination of conidia and spores[1]. This part has been used in the generation of transgenic lines of Tobacco plants which were resistant to <i>Alternaria, Phytophthora, Fusarium, Pythium</i>  fungi [2].</p>
  
 
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<span class='h3bb'>Sequence and Features</span>
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===<span class='h3bb'><b>Sequence and Features</b></span>===
 
<partinfo>BBa_K4147002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4147002 SequenceAndFeatures</partinfo>
  
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Before moving on to the experimental process, we first performed an <i>in silico</i> analysis in SnapGene®️ to simulate our ligated expression plasmid. The theoretical result was a sequence of 3,283 bp, as shown in <b>Figure 1.</b></p> 
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<center>[[File:Expression_construct_for_(CBD)2-DrsB1_sequence_7.png |300px]]</center>
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<center><b>Figure 1</b>. SnapGene®️ map of BioBrick BBa_K4147002.</center>
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<p align="justify">
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Our insert of PcOSM Construct with LacI regulated promoter was later synthetized by Twist Bioscience with the Biobrick prefix and suffix as well as adapters flanking the composite part for easiest restriction digest. EcoRI and PstI enzymes were used to digest both construct and vector. Once digested we proceed to ligate our insert into a pTwist Kan High Copy plasmid with Anza™ T4 DNA Ligase Master Mix. The ligation product was then transformed into <i>E. coli</i> BL21(DE3) cells by heat shock.</p>
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<p align="justify">
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The next step was to confirm the presence of the vector of interest in our chassis after transformation, so we performed colony PCR using Forward: 5'-GTTTCTTCGAATTCGCGGCCGCTTCTA and Reverse: 5'-GTTTCTTCCTTCCTGCAGCGGCCGCTACTAG primers specific for the BioBrick prefix and suffix respectively. The PCR action from SnapGene®️ was used to predict the resultant agarose gel.</p>
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<center>[[File:Agarose-gel-(CBD)2-DrsB1.png |400px]]</center>
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<center><b>Figure 2</b>.<i>Left.</i> SnapGene®️ amplification through PCR of BBa_K4147002 on an 0.8% agarose gel with NEB Quick-Load Purple 1Kb Plus DNA Ladder. <i>Right</i> C) Amplification of PcOSM Construct with LacI regulated promoter where the highlighted band corresponds to approximately 1,134 bp as expected.</center>
  
 
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===REFERENCES===
 
===REFERENCES===
 
[1] Shams, M. V., Nazarian-Firouzabadi, F., Ismaili, A., & Shirzadian-Khorramabad, R. (2019). Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity. Molecular Biotechnology. doi:10.1007/s12033-019-00153-x  
 
[1] Shams, M. V., Nazarian-Firouzabadi, F., Ismaili, A., & Shirzadian-Khorramabad, R. (2019). Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity. Molecular Biotechnology. doi:10.1007/s12033-019-00153-x  
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[2] Khademi, M., Varasteh-Shams, M., Nazarian-Firouzabadi, F., & Ismaili, A. (2020). New Recombinant Antimicrobial Peptides Confer Resistance to Fungal Pathogens in Tobacco Plants. Frontiers in Plant Science, 11. doi:10.3389/fpls.2020.01236
 
[2] Khademi, M., Varasteh-Shams, M., Nazarian-Firouzabadi, F., & Ismaili, A. (2020). New Recombinant Antimicrobial Peptides Confer Resistance to Fungal Pathogens in Tobacco Plants. Frontiers in Plant Science, 11. doi:10.3389/fpls.2020.01236

Latest revision as of 11:37, 12 October 2022


Expression construct for (CBD)2-DrsB1: Dermaseptin AMP from Phyllomedusa bicolor

This part contains the linear construct BBa_K4147001 for (CBD)2-DrsB1 which already incorporates a PelB signal peptide on the N- terminal as well as a 6X-His tag at the end of the dermaseptin CDS (C- terminal) for later protein purification. This composite construct includes a LacI regulated promoter. Additionally, it contains an RBS under the part name BBa_B0032. Finally, it has a double terminator (BBa_B0015) to ensure correct termination. As a linear 1075 bp construct, it contains the RFC10 prefix and suffix, making the part compatible with other iGEM parts.


Usage and Biology

Among a wide range of AMPs, polycationic dermaseptin peptides are produced by the skin glands of the frog Phyllomedusa bicolor. Although most dermaseptins are known to disrupt pathogens' lipid membranes, which leads to the development of membrane diseases, some may breach the membrane and interfere with critical cell components like DNA [1]. This system makes this AMPs the alternative antibiotics to boost plant innate immune system. It supplies both antibacterial and antifungal protection to a broad range of plant pathogens but shows no toxic effects on plant and mammalian cells [2]. This peptide of molecular weight of 22.1 kDa contains a tandem repeat of a chitin binding domain (CBD) as an improvement of dermaseptin-b1 gene (AKA MsrA2)(BBa_K2577001) which binds to chitin fungal cell wall, thereby increasing the lytic activity of the catalytic domain.

This fusion of DrsB1 to CBD not only protect DrsB1 from host degradation but its affinity for fungal cell wall chitin remains the same. It has been shown showed that this recombinant peptide possessed strong antifungal activity in vitro, inhibiting the growth of mycelia as well as the germination of conidia and spores[1]. This part has been used in the generation of transgenic lines of Tobacco plants which were resistant to Alternaria, Phytophthora, Fusarium, Pythium fungi [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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 392
    Illegal AgeI site found at 671
  • 1000
    COMPATIBLE WITH RFC[1000]


Before moving on to the experimental process, we first performed an in silico analysis in SnapGene®️ to simulate our ligated expression plasmid. The theoretical result was a sequence of 3,283 bp, as shown in Figure 1.

Expression construct for (CBD)2-DrsB1 sequence 7.png
Figure 1. SnapGene®️ map of BioBrick BBa_K4147002.

Our insert of PcOSM Construct with LacI regulated promoter was later synthetized by Twist Bioscience with the Biobrick prefix and suffix as well as adapters flanking the composite part for easiest restriction digest. EcoRI and PstI enzymes were used to digest both construct and vector. Once digested we proceed to ligate our insert into a pTwist Kan High Copy plasmid with Anza™ T4 DNA Ligase Master Mix. The ligation product was then transformed into E. coli BL21(DE3) cells by heat shock.

The next step was to confirm the presence of the vector of interest in our chassis after transformation, so we performed colony PCR using Forward: 5'-GTTTCTTCGAATTCGCGGCCGCTTCTA and Reverse: 5'-GTTTCTTCCTTCCTGCAGCGGCCGCTACTAG primers specific for the BioBrick prefix and suffix respectively. The PCR action from SnapGene®️ was used to predict the resultant agarose gel.

Agarose-gel-(CBD)2-DrsB1.png
Figure 2.Left. SnapGene®️ amplification through PCR of BBa_K4147002 on an 0.8% agarose gel with NEB Quick-Load Purple 1Kb Plus DNA Ladder. Right C) Amplification of PcOSM Construct with LacI regulated promoter where the highlighted band corresponds to approximately 1,134 bp as expected.


REFERENCES

[1] Shams, M. V., Nazarian-Firouzabadi, F., Ismaili, A., & Shirzadian-Khorramabad, R. (2019). Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity. Molecular Biotechnology. doi:10.1007/s12033-019-00153-x

[2] Khademi, M., Varasteh-Shams, M., Nazarian-Firouzabadi, F., & Ismaili, A. (2020). New Recombinant Antimicrobial Peptides Confer Resistance to Fungal Pathogens in Tobacco Plants. Frontiers in Plant Science, 11. doi:10.3389/fpls.2020.01236