Difference between revisions of "Part:BBa K3598010"
 
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Human Cathelicidin LL-37 is antimicrobial peptides produced by human in response to various pathogenic microbes. It has been shown to possess potent antimicrobial activity against a variety of bacteria.
 
Human Cathelicidin LL-37 is antimicrobial peptides produced by human in response to various pathogenic microbes. It has been shown to possess potent antimicrobial activity against a variety of bacteria.
  
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===Usage and Biology===
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[[File:T--BEIJING 4ELEVEN--Human_1.png|400px|thumb|center|Figure 1. Part demonstration]]
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[[File:T--BEIJING 4ELEVEN--Human_1.png|600px|thumb|center|Figure 1. Part demonstration]]
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The Human cathelicidin LL-37 coding part was the original sequence of Human cathelicidin LL-37 we sent to a synthetic company to produce our Human cathelicidin LL-37 AMP. We verified the antimicrobial potency of Human cathelicidin LL-37 purchased from the company by adding its solution and placing pieces of soaked with its solution to plates inoculated with P. acnes and E. coli MG1655. The results showed inhibition zones in areas of direct contact between bacteria and Human cathelicidin LL-37, which means Human cathelicidin LL-37 is effective in killing both bacteria, and would not affect bacteria outside its contact.  
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===Experiments and Results===
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[[File:T--BEIJING 4ELEVEN--Human_2|600px|thumb|center|Figure 2. Plate verification result of Human cathelicidin LL-37's ability to kill E. coli]]
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[[File:T--BEIJING 4ELEVEN--Human_3.png|600px|thumb|center|Figure 3. Plate verification result of Human cathelicidin LL-37's ability to kill P. acnes]]
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The part is the original sequence of Human Cathelicidin LL-37, we verified its antimicrobial potency through two methods: plate inhibition zones assay and OD600 analysis. In plate inhibition zones assay,wo placed pieces of filter paper soaked with AMP solutions onto plates inoculated with E.coli MG1655 and P.acnes and observing bacteria growth inhibition zones. In OD600 analysis, we added AMP solutions into liquid culture inoculated with the bacteria and measuring OD600 value.
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Then, we verified Human cathelicidin LL-37's ability to kill E. coli MG1655 and P. acnes by adding its solution to liquid culture inoculated with the two bacteria. We set the AMP concentration gradient as 0, 2, 4, 8, 12, 16 mg/L, and measured the OD600 of the E. coli through time. Since P. acnes has a slow growth rate, we only measured OD600 after 24 hours of cultivation. The results indicate that Snake Cathelicidin-BF's ability to kill E. coli MG1655 peaked at concentration 4mg/L at 8h, its effect reaching an OD600 of 2.0. Human cathelicidin LL-37's ability to kill P. acnes peaked at concentration 16mg/L, its effect reaching an OD600 of about 0.15.
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[[File:T--BEIJING 4ELEVEN--Human_4.png|600px|thumb|center|Figure 4. OD600 verification result of Human cathelicidin LL-37's ability to kill E. coli]]
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[[File:T--BEIJING_4ELEVEN--Collection_Figure_2.png|400px|thumb|center|Figure 2. AMPs test methods]]
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[[File:T--BEIJING 4ELEVEN--Human_5.png|600px|thumb|center|Figure 5. OD600 verification result of Human cathelicidin LL-37's ability to kill P. acnes]]
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====Plate verification Results====
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From the plates showed in Figure 3& 4,we can easily observe the inhibition zones in areas of direct contact between bacteria and Human Cathelicidin LL-37, while no inhibition zone formed around the filter paper. These results indicate that Human Cathelicidin LL-37 is effective to both E.coli and P.acnes, while would not affect bacteria outside its contact. Additionally, the edge of inhibition zone in E.coli plates wasn't clear, some colonies could survive around it, which means the AMP efficiency to E.coli is not as effective as P.acnes.
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[[File:T--BEIJING 4ELEVEN--010E.png|400px|thumb|center|Figure 3. Plate verification result of Human cathelicidin LL-37's efficiency to E. coli]]
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[[File:T--BEIJING 4ELEVEN--Human_3.png|400px|thumb|center|Figure 4. Plate verification result of Human cathelicidin LL-37's efficiency to kill P. acnes]]
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====OD600 Verification====
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Then, we verified Human cathelicidin LL-37's ability to kill E. coli MG1655 and P. acnes by adding its solution to liquid culture inoculated with the two bacteria. We set the AMP concentration gradient as 0, 2, 4, 8, 12, 16 mg/L, and measured the OD600 of the E. coli through time. Since P. acnes has a slow growth rate, we only measured OD600 after 48 hours of cultivation.
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 +
[[File:T--BEIJING 4ELEVEN--Human_4.png|400px|thumb|center|Figure 4. OD600 verification result of Human cathelicidin LL-37's ability to kill E. coli]]
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The results in Figure 4 confirmed our speculation in the plate tesst. The effect of this AMP to E. coli is not particularly good, its inhibition to the bacteria still in the early stage of cell growth even if the concentration rises to 16 mg/mL, and the final OD600 values of the experimental group and the control group's were almost the same.
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 +
[[File:T--BEIJING 4ELEVEN--0105.png|400px|thumb|center|Figure 5. OD600 verification result of Human cathelicidin LL-37's ability to kill P. acnes]]
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Although Human cathelicidin LL-37 is not very effective against E. coli, it exhibits strong effect on P.acnes. From the 48 h histogram (Figure 5),efficiency of Human cathelicidin LL-37 is obvious. At the concentration of 16 mg/mL, the OD600 vallue of the control group was 0.27, while the experimental group was only 0.16.
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Latest revision as of 13:47, 26 October 2020


Human CathelicidinLL-37

Human Cathelicidin LL-37 is antimicrobial peptides produced by human in response to various pathogenic microbes. It has been shown to possess potent antimicrobial activity against a variety of bacteria.

Figure 1. Part demonstration

Experiments and Results

The part is the original sequence of Human Cathelicidin LL-37, we verified its antimicrobial potency through two methods: plate inhibition zones assay and OD600 analysis. In plate inhibition zones assay,wo placed pieces of filter paper soaked with AMP solutions onto plates inoculated with E.coli MG1655 and P.acnes and observing bacteria growth inhibition zones. In OD600 analysis, we added AMP solutions into liquid culture inoculated with the bacteria and measuring OD600 value.

Figure 2. AMPs test methods

Plate verification Results

From the plates showed in Figure 3& 4,we can easily observe the inhibition zones in areas of direct contact between bacteria and Human Cathelicidin LL-37, while no inhibition zone formed around the filter paper. These results indicate that Human Cathelicidin LL-37 is effective to both E.coli and P.acnes, while would not affect bacteria outside its contact. Additionally, the edge of inhibition zone in E.coli plates wasn't clear, some colonies could survive around it, which means the AMP efficiency to E.coli is not as effective as P.acnes.

Figure 3. Plate verification result of Human cathelicidin LL-37's efficiency to E. coli
Figure 4. Plate verification result of Human cathelicidin LL-37's efficiency to kill P. acnes

OD600 Verification

Then, we verified Human cathelicidin LL-37's ability to kill E. coli MG1655 and P. acnes by adding its solution to liquid culture inoculated with the two bacteria. We set the AMP concentration gradient as 0, 2, 4, 8, 12, 16 mg/L, and measured the OD600 of the E. coli through time. Since P. acnes has a slow growth rate, we only measured OD600 after 48 hours of cultivation.

Figure 4. OD600 verification result of Human cathelicidin LL-37's ability to kill E. coli

The results in Figure 4 confirmed our speculation in the plate tesst. The effect of this AMP to E. coli is not particularly good, its inhibition to the bacteria still in the early stage of cell growth even if the concentration rises to 16 mg/mL, and the final OD600 values of the experimental group and the control group's were almost the same.

Figure 5. OD600 verification result of Human cathelicidin LL-37's ability to kill P. acnes

Although Human cathelicidin LL-37 is not very effective against E. coli, it exhibits strong effect on P.acnes. From the 48 h histogram (Figure 5),efficiency of Human cathelicidin LL-37 is obvious. At the concentration of 16 mg/mL, the OD600 vallue of the control group was 0.27, while the experimental group was only 0.16.


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]