Difference between revisions of "Part:BBa K2548013"
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This design is used to synthesize and extract distinct scydonadin in order to test its effectiveness. We chose the pTac promoter, a hybrid of Lac and Trp promoters, to induce scydonadin 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 sydonadin to extract it out with the nickel column.
 
This design is used to synthesize and extract distinct scydonadin in order to test its effectiveness. We chose the pTac promoter, a hybrid of Lac and Trp promoters, to induce scydonadin 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 sydonadin to extract it out with the nickel column.
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<h2>Construction of the device</h2>
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https://static.igem.org/mediawiki/parts/thumb/d/d5/SCY.png/800px-SCY.png
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<h2>Characterization</h2>
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<p>We examined the length of protein we extracted by performing SDS-PAGE to the supernatant after ultrasonic lysis. The protein size of SCY is 12.54 kDa. Protein bands 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 SCY was expressed. </p>
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        <div align="center"><img width="50%" src="https://static.igem.org/mediawiki/2018/2/2d/T--BNDS_CHINA--gel.png"/><br/>
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                Fig 9. Protein bands of antimicrobial peptides</div>
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<p>We tested the effect of different antimicrobial peptides through the procedures described in Experiments. The photos below show Aeromonas hydrophila under electron microscope. </p>
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        <div align="center"><img width="60%" src="https://static.igem.org/mediawiki/2018/9/95/T--BNDS_CHINA--SEM.png"/><br/>
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                Figure 10. Cells of A.hydrophila in control group</div>
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<p>Figure 10 shows a group of A.hydrophila in control group with smooth surface. The entire cell of A.hydrophila is lighter than that of the background.  </p>
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<div align="center"><img width="60%" src="https://static.igem.org/mediawiki/2018/c/c1/T--BNDS_CHINA--sem2.jpg"/><br/>
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              Figure 11: A cell of A.hydrophila processed by spALF4</div>
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<div align="center"><img width="60%" src="https://static.igem.org/mediawiki/2018/e/e3/T--BNDS_CHINA--sem3.jpg"/><br/>
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              Figure 12. Cells of A.hydrophila processed by SCY</div>
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<div align="center"><img width="60%" src="https://static.igem.org/mediawiki/2018/5/5b/T--BNDS_China--Sem.jpeg"/><br/>
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Figure 13. Cells of A.hydrophila processed by cPcAMP1</div>
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<p>Figure 10 to figure 13 show a group of broken A.hydrophila that has been processed by AMPs. 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. </p>
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<p>Through the electron microscope photos, we could demonstrate that our AMP solution caused damage on cell walls of A.hydrophila. </p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 21:44, 17 October 2018


LacI - pTac - Scygonadin + 6xHis Tag - Terminator

This design is used to synthesize and extract distinct scydonadin in order to test its effectiveness. We chose the pTac promoter, a hybrid of Lac and Trp promoters, to induce scydonadin 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 sydonadin to extract it out with the nickel column.

Construction of the device

800px-SCY.png

Characterization

We examined the length of protein we extracted by performing SDS-PAGE to the supernatant after ultrasonic lysis. The protein size of SCY is 12.54 kDa. Protein bands 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 SCY was expressed.

<img width="50%" src="T--BNDS_CHINA--gel.png"/>
Fig 9. Protein bands of antimicrobial peptides

We tested the effect of different antimicrobial peptides through the procedures described in Experiments. The photos below show Aeromonas hydrophila under electron microscope.

<img width="60%" src="T--BNDS_CHINA--SEM.png"/>
Figure 10. Cells of A.hydrophila in control group

Figure 10 shows a group of A.hydrophila in control group with smooth surface. The entire cell of A.hydrophila is lighter than that of the background.


<img width="60%" src="T--BNDS_CHINA--sem2.jpg"/>
Figure 11: A cell of A.hydrophila processed by spALF4
<img width="60%" src="T--BNDS_CHINA--sem3.jpg"/>
Figure 12. Cells of A.hydrophila processed by SCY
<img width="60%" src="T--BNDS_China--Sem.jpeg"/>
Figure 13. Cells of A.hydrophila processed by cPcAMP1

Figure 10 to figure 13 show a group of broken A.hydrophila that has been processed by AMPs. 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 AMP solution caused damage on cell walls of A.hydrophila.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1442
  • 1000
    COMPATIBLE WITH RFC[1000]