[[File:T--BEIJING 4ELEVEN--1. Lark20201024-223420.png|600px|thumb|center|Figure 1. Part demonstration]]
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The AOX1-Snake cathelicidin BF is a composite part consisting of an AOX1 promoter, a Snake cathelicidin BF sequence, and an AOX1 terminator. It is designed for the expression of our AMP Snake cathelicidin BF. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression.
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===Experiments and Results===
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We tested the antimicrobial potency of Snake cathelicidin BF as fermentation product of our Pichia pastoris by adding its solution to plates inoculated with P. acnes and E. coli MG1655. It can be inferred from the results that the fermentation product's effect of killing E. coli MG1655 and P. acnes are not significant. Only a certain amount of bacteria within contact of the products were eliminated and the inhibition zones are quite unclear due to low volume of AMPs dissolved in solutions.
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[[File:T--BEIJING 4ELEVEN--2-049.png|600px|thumb|center|Figure 2. Plate verification of Snake cathelicidin BF on P. acnes]]
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[[File:T--BEIJING 4ELEVEN--3-49.png|600px|thumb|center|Figure 3. Plate verification of Snake cathelicidin BF on E. coli]]
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Then, we verified Snake cathelicidin BF's ability as a fermentation product 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 2.5% and 25% in verification with E. coli and 25% with P. acnes because 2.5% proved in prior verification to be too low a concentration for the effects to be significant, and measured the OD600 of the bacteria. The results show that the fermentation product is effective in killing E. coli MG1655 and P. acnes.
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[[File:T--BEIJING 4ELEVEN--4.MG1655.png|600px|thumb|center|Figure 4. OD600 verification of 2.5% fermentation broth Snake cathelicidin BF on E. coli ]]
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[[File:T--BEIJING 4ELEVEN--5Snake Cathelicidin-BF Efficiency to MG1655 of 25% Fermentation Broth (2).png|600px|thumb|center|Figure 5. OD600 verification of 25% fermentation broth Snake cathelicidin BF on E. coli ]]
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[[File:TT--BEIJING 4ELEVEN--6Snake Cathelicidin-BF Efficiency to P.acnes .png|600px|thumb|center|Figure 6. OD600 verification of 25% fermentation broth Snake cathelicidin BF on P.acnes ]]
The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br.
Usage and Biology
Figure 1. Part demonstration
The AOX1-CEN1HC-Br is a composite part consisting of an AOX1 promoter, a CEN1HC-Br sequence, and an AOX1 terminator. It is designed for the expression of our AMP CEN1HC-Br. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression.
We tested the antimicrobial potency of CEN1HC-Br as fermentation product of our Pichia pastoris by adding its solution to plates inoculated with P. acnes and E. coli MG1655. It can be inferred from the results that the fermentation product's effect of killing E. coli MG1655 and P. acnes are not significant. Only a certain amount of bacteria within contact of the products were eliminated and the inhibition zones are quite unclear due to low volume of AMPs dissolved in solutions.
Figure 2. Plate verification of CEN1HC-Br on P. acnes
Figure 3. Plate verification of CEN1HC-Br on E. coli
Then, we verified CEN1HC-Br's ability as a fermentation product 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 2.5% and 25% in verification with E. coli and 25% with P. acnes because 2.5% proved in prior verification to be too low a concentration for the effects to be significant, and measured the OD600 of the bacteria. The results show that the fermentation product is effective in killing E. coli MG1655 and P. acnes.
Figure 4. OD600 verification of 2.5% fermentation broth CEN1HC-Br on E. coli
Figure 5. OD600 verification of 25% fermentation broth CEN1HC-Br on E. coli
Figure 6. OD600 verification of 25% fermentation broth CEN1HC-Br on P.acnes
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1311
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 937 Illegal XhoI site found at 1191
