Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Results"

Revision as of 13:38, 18 September 2015

NTU-LIHPAO-Taiwan

Design
Reference
- Reference

CPP-PYY

Fusion protein and test part

1. Plasmid construction

Signal peptide-PYY-His and His-PYY

We used EcoRI and PstI to digest our inserts and vectors to produce stick ends, and combined them to make recombinant plasmids. After construction of the plasmids, we used EcoRI and PstI and electrophoresis to check the validity.

[Fig.1-1] Electrophoresis result [Full Size]

After digested with EcoRI and PstI, the plasmids were smaller than that were digested with EcoRI. This result show the construction is successful.

TAT-PYY

[Fig.1-2] Electrophoresis result [Full Size]

After digested with EcoRI and PstI, the plasmids were smaller than that digested with EcoRI, and there were bands below, which were our inserts (TAT-PYY). The result show the combination of TAT-PYY DNA sequence and pSB1C3 is completed.

Y2R

[Fig.1-3] Electrophoresis result [Full Size]

After digested with EcoRI and PstI, there were two kinds of bands which were our vectors (upper bands, pcDNA 3.0) and inserts (lower bands, Y2R). The result mean the construction is successful.

2. Protein expression

Signal peptide-PYY-His and negative control

[Fig.1-4] Western blot result [Full Size]

We transformed plasmid into BL21 competent cell, extracted total protein, and used western blot to check whether the target protein was overexpressed.

We used antibody which would bind His-tag, so the band should appear when target protein perform.

But there are similar bands in both SP and negative control, which mean there are non-specific binding between antibody and other protein, and our target protein didn’t overexpress.

Nisin Selection

Nisin Immunity Test

1. 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.

2. 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

Suicide Kill Part

Plasmid construct

2015/08/14

[1] Gel electrophoresis

[Fig.3-1] pC1-RBS-NucA-Ter-pSB1C3(nucA) [Full Size]

[2] NucA(B) SEQUENCING

[Fig.3-2] NucA(B) SEQUENCING [Full Size]

There is no consistence between our gene and sequencing.

2015/08/19

[1] Gel electrophoresis

[Fig.3-3] pC1-RBS-NucA-Ter-pSB1C3(nucA) [Full Size]

[2] NucA(C) SEQUENCING

[Fig.3-4] NucA(C) SEQUENCING [Full Size]

There is no consistence between our gene and sequencing.

2015/08/24

[1] Gel electrophoresis

[Fig.3-5] pC1-RBS-NucA-Ter-pSB1C3(nucA) [Full Size]

Result

From the test 1 and 2, we proof that nuclease A have its function and it can cut the genomic DNA and plasmid DNA, because we found that the sequencing result have no consistence with our gene sequence. From the test 3, we can assure that nuclease A can really cut the DNA because we acquire a lot of weird band in the gel electrophoresis. Maybe the band we get is the genomic DNA which is cut by nuclease A.

E. coli growth curve

Furthermore, we compare the wild type E. coli and the E. coli transformed with nucA , that cause the cell death.

[Fig.3-6] E. coli growth curve [Full Size]

[Fig.3-7] nucA growth curve [Full Size]

From the growth curve, we find a evident difference between the empty plasmid and the suicide plasmid. We can proof the function of the nucA.

Suicide Sence Part

For L. casei

[1] Gel electrophoresis

[Fig.3-8] plac-RBS-CI-Ter-pSB1C3(Plac) [Full Size]

[2] Transformation

[Fig.3-9] Transformation [Full Size]

From the gel electrophoresis, we make sure the plasmid we construct in the E. coli is correct. However, our transformation in the L. casei always failed in all kinds of electroporation voltage and time.

For E. coli

[1] Gel electrophoresis

[Fig.3-10] plac-RBS-CI-Ter-pSB1C3(PlacE) [Full Size]

It should have a 3K band in A1 B1 C1, but we found that there are 2k band. Maybe our ligation is not successful. Also we can not find 3K band in A1 B1.

[2] Gel electrophoresis

[Fig.3-11] plac-RBS-CI-Ter-pSB1C3(PlacE) [Full Size]

We can not find any band in A, B, C so we presume that its ligation failed. In sample D, E, F we found that there are no 2K and 1K band. Therefore, we think ligation failed.

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 After digested with <i>EcoRI</i> and <i>PstI</i>, there were two kinds of bands which were our vectors (upper bands, pcDNA 3.0) and inserts (lower bands, Y2R). The result mean the construction is successful.
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 .&nbsp;&nbsp;Protein expression