Team:NTU-LIHPAO-Taiwan/Results

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  • CPP-PYY Fusion Protein Design
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CPP-PYY

CPP-PYY Fusion Protein Design

A fusion protein that combines the main product peptide YY with signaling peptide, cell penetrating peptide and a thrombin-cleavable linker is designed. These additional parts all have individual functions.

[Fig.1-1] Healthin Design

Nisin Selection

Nisin Immunity Test

Nisin sensitivity of E. coli transformed with the plasmid expressing nisI in different concentration of nisin

The nisin sensitivity of E. coli was tested using agar diffusion method. Overnight cell culture carrying nisI gene was intensely streaked on LB agar plates. Discs containing different concentration of nisin were also put on the plates to examine the sensitivity. The plates were incubated at 37℃ overnight and the diameter of the inhibition zones were measured.

[Fig.2-1] Healthin Design [Full Size]

[Fig.2-2] Healthin Design [Full Size]

The result shows that in the low concentration of the nisin, E. coli has the immunity towards nisin. From the pictures shown above, there are no evident zones of inhibition in 0, 20, 50 IU. Therefore, we can assure that E. coli can expel nisin out of the cell body and survive well.

Nisin sensitivity of E. coli which cell wall is weakened by EDTA and transformed with the plasmid expressing nisI in different concentrations of nisin

Overnight E. coli cell culture was refreshed to O.D.₆₀₀ ~ 0.1 and then the LB broth was replaced by cell buffer containing 40 mM of EDTA. 100 µL of cell culture was mixed with the same volume of nisin solution of various concentrations and incubated at 37℃ for 1 hour. After that, nisin solution was removed by centrifuge. The cell culture that went through serial dilution to 10^-4 was dispersed on LB agar plates and incubated at 37℃ overnight to calculate the living amount of bacteria.

[Fig.2-3] Healthin Design [Full Size]

When treated with EDTA, a chelate that can weaken the outer membrane of E. coli, nisin was more likely to enter the cell body. Furthermore, we can easily deduce the factor, like cell wall thickness that influences the ability of nisin resistancy. From the result above, we can see that the higher concentration of nisin solution, the less the bacterial colonies, which indicates that the gene nisI help E. coli to get the ability to resist nisin.

Suicide

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