Difference between revisions of "Part:BBa K3733007"
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===Materials and Method===
 
===Materials and Method===
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<p>1.Plasmids Construction</p>
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<p>1.Plasmids Construction: LTA-coding sequence is constructed using primers by overlap extension PCR. The constructed fragment is ligated to the target site in pET28 by PCR and homologous recombination. The correct construction is confirmed by sequencing.</p>
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<p><br></br></p>
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<p><br></p>
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<p>LTA-coding sequence is constructed using primers by overlap extension PCR. The constructed fragment is ligated to the target site in pET28 by PCR and homologous recombination. The correct construction is confirmed by sequencing.</p>
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<p>2.Expression and Purification</p>
 +
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Plasmid pET-28a(+)-LTA (with His-tag) is transformed to <i>Escherichia coli</i> BL21(DE3). The E. coli strain is cultured in LB medium containing 10 μg/mL kanamycin.</p>
  
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<p>&nbsp;&nbsp;&nbsp;&nbsp;2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 50mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (<b>Sangon Biotech</b>, Shanghai, China).</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;3) <i>Salmonella Typhimurium</i> SL1344 is cultured in LB medium at 37℃ overnight. Resuspend the cultured <i>S. Typhimurium SL1344</i> by PBS. All activity tests are implemented in PBS resuspended <i>S. Typhimurium</i> SL1344. Culture the PBS resuspended <i>S. Typhimurium</i> SL1344 to OD<sub>600</sub> = 0.35.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;4) In the 96-well plates, 20 μl LTA solution is added to the final concentration of 8 μg/mL with 180 μl bacteria suspension in each well. Together with 20 μl Elution buffer (<b>Sangon Biotech</b>, Shanghai, China) added with 180 μl bacteria suspension serve as control groups. Each with three biological repeats.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Use the Microplate Reader to measure OD<sub>600</sub> of the 96-well-plate for 2.5 hours.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;6) All experiments above are carried out in 3 biological replications.</p>
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===Results===
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<center><img src="https://static.igem.org/mediawiki/parts/e/e9/T--HZAU-China--BBa_K3733007_img1.png" style="width:40%"></center>
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<center><b>Figure 1.</b> Tricine-SDS-PAGE analysis of LTA</center>
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<br>
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</body>
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</html>
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<p>The molecular weight of LTA is around 4.5kDa. There is a clear band between the 4.1kDa and 6.5kDa. Thus, the result of Tricine-SDS-PAGE above are the protein which has undergone affinity chromatography, indicating that there do have the expression of LL-37 in our chassis <i>Escherichia coli</i> BL21(DE3).</p>
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<center><img src="https://static.igem.org/mediawiki/parts/d/de/T--HZAU-China--BBa_K3733007_img2.png" style="width:40%"></center>
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<center><b>Figure 2.</b> OD<sub>600</sub>-Time curve of <i>Salmonella Typhimurium</i> SL1344 in the presence or absence of LTA.</center>
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<br>
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</body>
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<p>Compared with the control groups, LTA do have antibacterial effect with the OD<sub>600</sub> has an obvious lower increase. We successfully express and purify the LTA in the <i>Escherichia coli</i></p>.
  
 
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<partinfo>BBa_K3733007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3733007 SequenceAndFeatures</partinfo>
  
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===References===
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Zhang L, Wei X, Zhang R, et al. Design and development of a novel peptide for treating intestinal inflammation[J]. Frontiers in immunology, 2019, 10: 1841.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 14:20, 20 October 2021


LTA: A novel antimicrobial peptide

LTA is a novel antimicrobial peptide (AMP), whose design was based on combing the active centers of a ride range of AMPs, including LL-37, YW12D, innate defense regulator 1, and cathelicidin 2 with thymopentin or the active center of thymosin alpha 1 (Tα1). It could neutralize Lipopolysaccharides (LPS), thus effectively blocking the downstream inflammation pathway.


Design

This part has undergone codon optimization based on the bias of Escherichia coli. As the original coding sequence of LTA doesn’t contain the start codon and termination codon, they are then added to guarantee the expression of the protein.

Materials and Method

1.Plasmids Construction: LTA-coding sequence is constructed using primers by overlap extension PCR. The constructed fragment is ligated to the target site in pET28 by PCR and homologous recombination. The correct construction is confirmed by sequencing.


2.Expression and Purification

    1) Plasmid pET-28a(+)-LTA (with His-tag) is transformed to Escherichia coli BL21(DE3). The E. coli strain is cultured in LB medium containing 10 μg/mL kanamycin.

    2) When the optical density of the cultured bacteria reached approximately 0.6, IPTG was added to the final concentration 50mM. And the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China).

    3) Salmonella Typhimurium SL1344 is cultured in LB medium at 37℃ overnight. Resuspend the cultured S. Typhimurium SL1344 by PBS. All activity tests are implemented in PBS resuspended S. Typhimurium SL1344. Culture the PBS resuspended S. Typhimurium SL1344 to OD600 = 0.35.

    4) In the 96-well plates, 20 μl LTA solution is added to the final concentration of 8 μg/mL with 180 μl bacteria suspension in each well. Together with 20 μl Elution buffer (Sangon Biotech, Shanghai, China) added with 180 μl bacteria suspension serve as control groups. Each with three biological repeats.

    5) Use the Microplate Reader to measure OD600 of the 96-well-plate for 2.5 hours.

    6) All experiments above are carried out in 3 biological replications.

Results

无标题文档

Figure 1. Tricine-SDS-PAGE analysis of LTA

The molecular weight of LTA is around 4.5kDa. There is a clear band between the 4.1kDa and 6.5kDa. Thus, the result of Tricine-SDS-PAGE above are the protein which has undergone affinity chromatography, indicating that there do have the expression of LL-37 in our chassis Escherichia coli BL21(DE3).

无标题文档

Figure 2. OD600-Time curve of Salmonella Typhimurium SL1344 in the presence or absence of LTA.

Compared with the control groups, LTA do have antibacterial effect with the OD600 has an obvious lower increase. We successfully express and purify the LTA in the Escherichia coli

.

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]

References

Zhang L, Wei X, Zhang R, et al. Design and development of a novel peptide for treating intestinal inflammation[J]. Frontiers in immunology, 2019, 10: 1841.