Part:BBa_K2548012
LacI - pTac - SpALF4 + 6xHis Tag - Terminator
This design is used to synthesize and extract distinct SpALF4 in order to test its effectiveness. We chose the pTac promoter, a hybrid of Lac and Trp promoters, to induce SpALF4 production in E. coli because it is the most accessible promoter in our lab. The promoter can be induced by IPTG to remove the repression on transcription from LacI. We also add 6xHis-tag after sequences of SpALF4 to extract it out with the nickel column.
Construction of the device
Characterization
We examined the length of protein we extracted by performing SDS-PAGE to the supernatant after ultrasonic lysis. The protein size of SpALF4 is 11.54 kDa. Protein band in the correct molecular weight range were visualized in the area between 15 kDa and 10 kDa. There was no protein band in the same area for control group, indicating that SpALF4 was successfully expressed.
We tested the effect of different antimicrobial peptides through the procedures described in Experiments. The photo below show Aeromonas hydrophila under electron microscope.
Figure 2 shows a group of broken A.hydrophila that has been processed by SpALF4. The edges of the cells are clearly rougher than those of cells in the control group, with some obvious light leaves as circled in blue. We suppose the darkened parts among and besides the cells (circled in red) are the cell structures flowed out of the cell via the gaps on cell walls.
Through the electron microscope photos, we could demonstrate that our SpALF4 solution caused damage on cell walls of A.hydrophila.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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