Difference between revisions of "Part:BBa K3598051"

Latest revision as of 14:54, 27 October 2020

AOX1 Promoter_α factor secretion signal_Human CathelicidinLL-37_AOX 1 Terminator

The circuit we transformed into Pichia Pastoris to produce AMP Humancathelicidin-37.

Demonstration

Figure 1. Part demonstration

This part is a composite part consisting of an AOX1 promoter, a Human cathelicidin LL-37 sequence, and an AOX1 terminator. It is designed for the expression of our AMP Humancathelicidin-37. 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.

Experiments and Results

In our experiments, Humancathelicidin-37 was produced by fermentation in BMMY medium, 5% methanol was added every day to induce its expression. During the fermentation, we took sample every 24 h, and the supernatant was used to test the antibacterial effect for AMP was secreted extracellularly.

In the plate test, we add Humancathelicidin-37 fermentation supernatant directly to the plate with P. acnes and E. coli MG1655. From the results (Figure 2&3), we found the inhibition zone were not clear, which means their efficiency against bacteria are not obvious. That may be caused by the low concentration of antimicrobial peptides in the fermentation broth.

Figure 2. Plate verification of Human cathelicidin LL-37 on P. acnes

Figure 3. Plate verification of Human cathelicidin LL-37 on E. coli

Then, we verified Humancathelicidin-37's potency by adding the fermentation broth to liquid culture inoculated with the two E.coli MG1655 and P.acnes, 25% was added in verification to E. coli and 20% added to P. acnes. The OD600 were measured at 12 h for E.coli, while P.acnes was measured at 48h. The results were shown in Figure 5&6, from which we can infer that the fermentation product was effective in inhibting the growth of E. coli MG1655 and P. acnes.

Figure 5. OD600 verification of 25% fermentation broth Human cathelicidin LL-37 on E. coli

Figure 6. OD600 verification of 20% fermentation broth Human cathelicidin LL-37 on P.acnes

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1335
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 937
Illegal XhoI site found at 1191
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1401
1000
COMPATIBLE WITH RFC[1000]

@@ Line 10: / Line 10: @@
 [[File:T--BEIJING 4ELEVEN--1._Lark20201024-223452.png|400px|thumb|center|Figure 1. Part demonstration]]
-The AOX1-Human cathelicidin LL-37 is a composite part consisting of an AOX1 promoter, a Human cathelicidin LL-37 sequence, and an AOX1 terminator. It is designed for the expression of our AMP tredicapeptide. 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.
+This part is a composite part consisting of an AOX1 promoter, a Human cathelicidin LL-37 sequence, and an AOX1 terminator. It is designed for the expression of our AMP Humancathelicidin-37. 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 Human cathelicidin LL-37 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.
+===Experiments and Results===
+In our experiments, Humancathelicidin-37 was produced by fermentation in BMMY medium, 5% methanol was added every day to induce its expression. During the fermentation, we took sample every 24 h, and the supernatant was used to test the antibacterial effect for AMP was secreted extracellularly.
+In the plate test, we add Humancathelicidin-37 fermentation supernatant directly to the plate with P. acnes and E. coli MG1655. From the results (Figure 2&3), we found the inhibition zone were not clear, which means their efficiency against bacteria are not obvious. That may be caused by the low concentration of antimicrobial peptides in the fermentation broth.
 [[File:T--BEIJING 4ELEVEN--2.51.png|400px|thumb|center|Figure 2. Plate verification of Human cathelicidin LL-37 on P. acnes]]
@@ Line 18: / Line 23: @@
 [[File:T--BEIJING 4ELEVEN--3.51.png|400px|thumb|center|Figure 3. Plate verification of Human cathelicidin LL-37 on E. coli]]
-Then, we verified Human cathelicidin LL-37'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.
+Then, we verified Humancathelicidin-37's potency by adding the fermentation broth to liquid culture inoculated with the two E.coli MG1655 and P.acnes, 25% was added in verification to E. coli and 20% added to P. acnes. The OD600 were measured at 12 h for E.coli, while P.acnes was measured at 48h. The results were shown in Figure 5&6, from which we can infer that the fermentation product was effective in inhibting the growth of E. coli MG1655 and P. acnes.
 [[File:T--BEIJING 4ELEVEN--5.51.png|400px|thumb|center|Figure 5. OD600 verification of 25% fermentation broth Human cathelicidin LL-37 on E. coli]]