Difference between revisions of "Part:BBa K3838555"
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1. The DNA level
 
1. The DNA level
  
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We selected the plasmid PJL for verification and transformed it into DH5α (DJL) and Nissle 1917(NJL). The plasmid was extracted from the transformed  DH5a (DJL) , as shown in Figure 1A. The sample band was about 5000 bp and the target band size should be 4677 bp, which was considered to be the open loop conformation of the plasmid. In addition, the plasmid concentration extracted by Nissle was too low, so PCR validation was carried out on the plasmids in DJT and NJT, as shown in figure 1. The target band size was 945 bp, and the actual electrophoresi
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We selected the plasmid PJL for verification and transformed it into DH5α (DJL) and Nissle 1917(NJL). The plasmid was extracted from the transformed  DH5a (DJL) , as shown in Figure 1A. The sample band was about 5000 bp and the target band size should be 4677 bp, which was considered to be the open loop conformation of the plasmid. In addition, the plasmid concentration extracted by Nissle was too low, so PCR validation was carried out on the plasmids in DJT and NJT, as shown in figure 1. The target band size was 945 bp, and the actual electrophoresis results met the target band size.
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[[File:T--SZU-China--BBa K3838555-JLL371A.png|200px|center]]
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[[File:T--SZU-China--BBa K3838555-JLL371B.png|400px|center]]
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[[File:T--SZU-China--BBa K3838555-JLL371C.png|200px|center]]
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<center>Fig.1 A Plasmid was extracted from transformed DH5α and gel electrophoresis was performed. The target band size was 4677 bp. B PCR was performed on the plasmid extracted from transformed DH5α. C PCR was performed on the plasmid extracted from transformed Nissle 1917.</center>
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2. Protein level
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We validated the engineered bacteria at the protein level. As shown in Figure 28, for DH5α, a 4.5kDa LL37 protein band can be clearly seen in lane 4 compared with the LL37 standard in lane 2 and the control group in lane 5, as indicated by the arrow. The bands are clear and meet the expected size, which confirms the successful expression of our LL37 protein.
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[[File:T--SZU-China--resultLL37SDSPAGE.png|500px|center]]
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<center>Fig.2 SDS-PAGE of LL37 protein from engineered bacteria.</center>
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===References===
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[1]Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K, Roche FM, Mu R, Doho GH, Pistolic J, Powers JP, Bryan J, Brinkman FS, Hancock RE. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol. 2006 Feb 15;176(4):2455-64. doi: 10.4049/jimmunol.176.4.2455. PMID: 16456005.

Revision as of 14:44, 21 October 2021


J-LL37

LL37 is an antimicrobial peptide secreted by human immune cells, which can inhibit harmful bacteria such as Helicobacter pylori and Staphylococcus aureus. At concentrations usually found on adult mucosal surfaces (1-5 μg/ml), the peptide does not play a direct bactericidal role, but participates in immune regulation, and LL37 protects against endotoxemia/septicemia. At very low concentrations (≤1 μg/mL) and under physiological salt conditions in vivo, LL-37 has powerful anti-endotoxin properties and can regulate inflammatory response by inhibiting the release of TNF-α in LPS-stimulated human monocytes and reducing LPS-induced transport of P50 / P65 to the nucleus. LL37 inhibits LPS-induced gene transcription and plays an anti-endotoxin role. In the presence of LPS, LL37 significantly inhibited the expression of specific pro-inflammatory genes up-regulated by NF-κB. LL37 treatment also significantly changed the species and abundance of intestinal flora in mice, revealing its important effect on intestinal flora.

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]


Experience:SZU-China 2021 TEAM

1. The DNA level

We selected the plasmid PJL for verification and transformed it into DH5α (DJL) and Nissle 1917(NJL). The plasmid was extracted from the transformed DH5a (DJL) , as shown in Figure 1A. The sample band was about 5000 bp and the target band size should be 4677 bp, which was considered to be the open loop conformation of the plasmid. In addition, the plasmid concentration extracted by Nissle was too low, so PCR validation was carried out on the plasmids in DJT and NJT, as shown in figure 1. The target band size was 945 bp, and the actual electrophoresis results met the target band size.

T--SZU-China--BBa K3838555-JLL371A.png
T--SZU-China--BBa K3838555-JLL371B.png
T--SZU-China--BBa K3838555-JLL371C.png
Fig.1 A Plasmid was extracted from transformed DH5α and gel electrophoresis was performed. The target band size was 4677 bp. B PCR was performed on the plasmid extracted from transformed DH5α. C PCR was performed on the plasmid extracted from transformed Nissle 1917.


2. Protein level We validated the engineered bacteria at the protein level. As shown in Figure 28, for DH5α, a 4.5kDa LL37 protein band can be clearly seen in lane 4 compared with the LL37 standard in lane 2 and the control group in lane 5, as indicated by the arrow. The bands are clear and meet the expected size, which confirms the successful expression of our LL37 protein.

500px
Fig.2 SDS-PAGE of LL37 protein from engineered bacteria.

References

[1]Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K, Roche FM, Mu R, Doho GH, Pistolic J, Powers JP, Bryan J, Brinkman FS, Hancock RE. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol. 2006 Feb 15;176(4):2455-64. doi: 10.4049/jimmunol.176.4.2455. PMID: 16456005.