Difference between revisions of "Part:BBa K3351015"
 
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===Research===
 
===Research===
−
1.Positive colonies (E. coli BL21 (DE3) pWDX(+)::PhaP-HD5d5) from September 12, 2020 were transferred to test tubes with 2 mL LB+amp and incubated at 37°C, 200 rpm overnight. One 250 mL Erlenmeyer flask with 100 mL LB and 100µL amp/kan was inoculated with 1 mL of one culture above in a 250 mL flask, which both were incubated at 37°C, 200 rpm. OD measurement after three hours showed an OD(600) = 0.6. E. coli BL21 cultures were induced with 100 µL IPTG for incubated at 37°C, 200 rpm overnight.
+
1.Positive colonies (E. coli BL21 (DE3) pWDX(+)::PhaP-HD5d5) from September 12, 2020 were transferred to test tubes with 2 mL LB+amp and incubated at 37°C, 200 rpm overnight. One 250 mL Erlenmeyer flask with 100 mL LB and 100µL amp was inoculated with 1 mL of one culture above in a 250 mL flask, which both were incubated at 37°C, 200 rpm. OD measurement after three hours showed an OD(600) = 0.6. E. coli BL21 cultures were induced with 100 µL IPTG for incubated at 37°C, 200 rpm overnight.
 
The bacterial growth curves at OD600 were recorded at 30-min intervals for 15 hours.<br>
 
The bacterial growth curves at OD600 were recorded at 30-min intervals for 15 hours.<br>
 
<html>
 
<html>
 
<img style="display: block;
 
<img style="display: block;
−
     width: 60%;height: 60%;" src="https://2020.igem.org/wiki/images/0/0b/T--NWU-CHINA-A--p-hd5d5-01.png"><div>Figure.3  </div></html>
+
     width: 60%;height: 60%;" src="https://static.igem.org/mediawiki/parts/6/6f/T--NWU-CHINA-A--od-10.png"><div>Figure.3  </div></html>
 
After induction for 85~6 hours, the sludge was collected by centrifugation and analyzed by SDS-PAGE. The results showed that the antibacterial peptides fused with PhaP were all expressed, among which PhaP-HD5 and PhaP-HD5d5 expressed the most.
 
After induction for 85~6 hours, the sludge was collected by centrifugation and analyzed by SDS-PAGE. The results showed that the antibacterial peptides fused with PhaP were all expressed, among which PhaP-HD5 and PhaP-HD5d5 expressed the most.
 
<html>
 
<html>
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     width: 60%;height: 60%;" src="https://2020.igem.org/wiki/images/e/e6/T--NWU-CHINA-A--p-hd5-f4.png"><div>Figure.4  </div></html>
 
     width: 60%;height: 60%;" src="https://2020.igem.org/wiki/images/e/e6/T--NWU-CHINA-A--p-hd5-f4.png"><div>Figure.4  </div></html>
 
2.The incubated medium was transferred to 50 mL centrifuge tube and the supernatant collected after centrifuging (6791×g) for 3 min at 4°C. After removal of the supernatant by centrifugation (6791×g, 15 min), it was collected precipitate.<br>
 
2.The incubated medium was transferred to 50 mL centrifuge tube and the supernatant collected after centrifuging (6791×g) for 3 min at 4°C. After removal of the supernatant by centrifugation (6791×g, 15 min), it was collected precipitate.<br>
−
3.The bacteria were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 2 min. (A volume of 20 ml of the Bacterial lysates was added to each baterial precipitat, Bacterial lysates 2.0: 20mM/L tris-HCl  150mM/L NaCl)<br>
+
3.The bacteria were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 2 min. (A volume of 20 ml of the Bacterial lysis solution was added to each baterial precipitat, Bacterial lysis solution 2.0: 20mM/L tris-HCl  150mM/L NaCl)<br>
 
4.PhaP-HD5d5 was not observed in the supernatant analyzed by SDS-PAGE.<br>
 
4.PhaP-HD5d5 was not observed in the supernatant analyzed by SDS-PAGE.<br>
  
 
===Research and design cycle:===
 
===Research and design cycle:===
−
Through analysis, the reason we thought was the Bacterial lysates or the ultrasonic power/total time,so we change the Bacterial lysates and ultrasonic total time.<br>
+
Through analysis, the reason we thought was the Bacterial lysis solution or the ultrasonic power/total time,so we change the Bacterial lysis solution and ultrasonic total time.<br>
−
The bacterias were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 15 min. (A volume of 20 ml of the Bacterial lysates was added to each baterial precipitat, Bacterial lysates 2.0: 20mM/L tris-HCl  150mM/L NaCl). But PhaP-HD5d5 also was not observed in the supernatant analyzed by SDS-PAGE.<br>
+
1.The bacterias were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 15 min. (A volume of 20 ml of the Bacterial lysis solution was added to each baterial precipitat, Bacterial lysis solution 2.0: 20mM/L tris-HCl  150mM/L NaCl). But PhaP-HD5d5 also was not observed in the supernatant analyzed by SDS-PAGE.<br>
−
This time,we increased the power of ultrasound and the time to crush the bacteria,which were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 30 min.it is pity that we couldn’t observed the PhaP-HD5d5 in in the supernatant analyzed by SDS-PAGE.<br>
+
2.This time,we increased the power of ultrasound and the time to crush the bacteria,which were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 30 min.it is pity that we couldn’t observed the PhaP-HD5d5 in in the supernatant analyzed by SDS-PAGE.<br>
−
So we increased the total time of ultrasound to 45 min, the bacterias were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 45 min.The sonicated bacterial lysate was centrifuged 6791 × g for 15 min at 4°C. Equivalent amounts of supernatant and precipitate were subjected to SDS-PAGE analysis. The supernatant, containing target proteins(PhaP-HD5d5)<br>
+
3.So we increased the total time of ultrasound to 45 min, the bacteria were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 45 min. The sonicated bacterial lysis solution was centrifuged 6791 × g for 15 min at 4°C. Equivalent amounts of supernatant and precipitate were subjected to SDS-PAGE analysis. The supernatant, containing target proteins(PhaP-HD5d5)<br>
 
<html><img style="display: block;
 
<html><img style="display: block;
 
     width: 50%;height: 50%;" src="https://2020.igem.org/wiki/images/8/83/T--NWU-CHINA-A--p-hd5d5-gel.png"><div>Figure.5 </div></html>
 
     width: 50%;height: 50%;" src="https://2020.igem.org/wiki/images/8/83/T--NWU-CHINA-A--p-hd5d5-gel.png"><div>Figure.5 </div></html>
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<html>
 
<html>
 
<img style="display: block;
 
<img style="display: block;
−
     width: 60%;height: 60%;" src="https://2020.igem.org/wiki/images/0/0b/T--NWU-CHINA-A--p-hd5d5-01.png"><div>Figure.7</div></html>
+
     width: 60%;height: 60%;" src="https://static.igem.org/mediawiki/parts/6/6f/T--NWU-CHINA-A--od-10.png"><div>Figure.7</div></html>
 +
 
 +
In order to verify the adhesion between PHA and PhaP-AMPs ,we did the following experiments:<br>
 +
First ,we used electrospinning technology to make PHA films.<br>
 +
Secondly the films was immersed in the supernatant after bacterial sonication for 2 hours. <br>
 +
Thirdly, we washed the films gently with lysis buffer, then put them into a centrifuge tube and added SDS-PAGE buffer, heat at 90℃ until the thin membrane softens.<br>
 +
In order to make the adhesion between PhaP-AMPs and PHA better, we used nano-level PHA for verification. Added PHA and supernatant in a ratio of 1:20, stirred two hours by the magnetic stirrer, then centrifuged at 8000rpm for 2min, rinsed twice with lysate, and added SDS-PAGE buffer.<br>
 +
We took the liquid from the above product for SDS-PAGE, as we can see, PhaP-HD5d5 combines the most products with PHA，whether it is electrospinning or nanoparticles.<br>
 +
<html>
 +
<img style="display: block;
 +
    width: 80%;height: 80%;" src="https://static.igem.org/mediawiki/parts/e/e7/T--NWU-CHINA-A--gel3.png"><div>Figure.8</div></html>
  
 
===Reference===
 
===Reference===
−
[1] Xue Q, Liu XB, Lao YH, Wu LP, Wang D, Zuo ZQ, Chen JY, Hou J, Bei YY, Wu XF, Leong KW, Xiang H, Han J. Anti-infective biomaterials with surface-decorated tachyplesin I. Biomaterials. 2018 Sep;178:351-362. doi: 10.1016/j.biomaterials.2018.05.008. Epub 2018 May 9. PMID: 29778319.
+
[1] Xue Q, Liu XB, Lao YH, Wu LP, Wang D, Zuo ZQ, Chen JY, Hou J, Bei YY, Wu XF, Leong KW, Xiang H, Han J. Anti-infective biomaterials with surface-decorated tachyplesin I. Biomaterials. 2018 Sep;178:351-362. doi: 10.1016/j.biomaterials.2018.05.008. Epub 2018 May 9. PMID: 29778319.<br>
 +
[2] Wang, Cheng et al. “A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.” Antimicrobial agents and chemotherapy vol. 62,2 e01504-17. 25 Jan. 2018, doi:10.1128/AAC.01504-17
 +
 
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 08:13, 27 October 2020


HisTag-PhaP-linker-HD5d5-HisTag

Summary

PhaP contains a hydrophobic granule binding domain and a cytosol-facing hydrophilic domain. Complete biodegradable nature of PhaP. PhaP-tagged proteins could interact with various types of hydrophobic surfaces. A (Gly4Ser2)2 flexible linker is inserted between PhaP and HD5d5 to facilitate attaching of HD5d5 on surface and displaying sufficient flexibility of this peptide.
The noncationic and nonhydrophobic residues of HD5 were replaced with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb.

Figure.1

Imagine

The fusion protein PhaP-HD5d5 was expressed in E. coli strain Transetta (DE3).Because PhaP-tagged proteins could interact with various types of hydrophobic surfaces. PhaP-HD5d5 will anchor to hydrophobic polymer surface efficiently to make band-aids with antibacterial functions.

Design

1.HD5d5: Mining Gene Information from the Literature.
2.The HD5d5 fragment was subcloned into the pWDX plasmid using the HindⅢ and SpeⅠ sites to construct the pWDX-HD5d5 plasmid.

Figure.2
3.The plasmids were then transformed into E. coli BL21(DE3), and transformant clones were screened.
4.E. coli BL21 (DE3) was used as the protein expression host.
5.Extraction and Purification of Proteins (PhaP-HD5d5) from E. coli BL21 (DE3).
6.The in vitro antibacterial activities of PhaP-HD5d5 was measured against S. aureus using the filter paper disc diffusion method.
7.PhaP-HD5d5 will be adhered to the surface of PHA.
8.The bactericidal activity of PhaP, PhaP-HD5d5, and their coated PHA films will be tested against selected bacteria.

Research

1.Positive colonies (E. coli BL21 (DE3) pWDX(+)::PhaP-HD5d5) from September 12, 2020 were transferred to test tubes with 2 mL LB+amp and incubated at 37°C, 200 rpm overnight. One 250 mL Erlenmeyer flask with 100 mL LB and 100µL amp was inoculated with 1 mL of one culture above in a 250 mL flask, which both were incubated at 37°C, 200 rpm. OD measurement after three hours showed an OD(600) = 0.6. E. coli BL21 cultures were induced with 100 µL IPTG for incubated at 37°C, 200 rpm overnight. The bacterial growth curves at OD600 were recorded at 30-min intervals for 15 hours.

Figure.3
After induction for 85~6 hours, the sludge was collected by centrifugation and analyzed by SDS-PAGE. The results showed that the antibacterial peptides fused with PhaP were all expressed, among which PhaP-HD5 and PhaP-HD5d5 expressed the most.
Figure.4
2.The incubated medium was transferred to 50 mL centrifuge tube and the supernatant collected after centrifuging (6791×g) for 3 min at 4°C. After removal of the supernatant by centrifugation (6791×g, 15 min), it was collected precipitate.
3.The bacteria were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 2 min. (A volume of 20 ml of the Bacterial lysis solution was added to each baterial precipitat, Bacterial lysis solution 2.0: 20mM/L tris-HCl 150mM/L NaCl)
4.PhaP-HD5d5 was not observed in the supernatant analyzed by SDS-PAGE.

Research and design cycle:

Through analysis, the reason we thought was the Bacterial lysis solution or the ultrasonic power/total time,so we change the Bacterial lysis solution and ultrasonic total time.
1.The bacterias were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval time: 1s) on ice for 15 min. (A volume of 20 ml of the Bacterial lysis solution was added to each baterial precipitat, Bacterial lysis solution 2.0: 20mM/L tris-HCl 150mM/L NaCl). But PhaP-HD5d5 also was not observed in the supernatant analyzed by SDS-PAGE.
2.This time,we increased the power of ultrasound and the time to crush the bacteria,which were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 30 min.it is pity that we couldn’t observed the PhaP-HD5d5 in in the supernatant analyzed by SDS-PAGE.
3.So we increased the total time of ultrasound to 45 min, the bacteria were crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 45 min. The sonicated bacterial lysis solution was centrifuged 6791 × g for 15 min at 4°C. Equivalent amounts of supernatant and precipitate were subjected to SDS-PAGE analysis. The supernatant, containing target proteins(PhaP-HD5d5)

Figure.5

Bacteriostatic experimen

It was noticed that PhaP-HD5d5 showed the antibacterial effects when examined on S. aureus strain RN4220.

Figure.6


Characterization

Figure.7

In order to verify the adhesion between PHA and PhaP-AMPs ,we did the following experiments:
First ,we used electrospinning technology to make PHA films.
Secondly the films was immersed in the supernatant after bacterial sonication for 2 hours.
Thirdly, we washed the films gently with lysis buffer, then put them into a centrifuge tube and added SDS-PAGE buffer, heat at 90℃ until the thin membrane softens.
In order to make the adhesion between PhaP-AMPs and PHA better, we used nano-level PHA for verification. Added PHA and supernatant in a ratio of 1:20, stirred two hours by the magnetic stirrer, then centrifuged at 8000rpm for 2min, rinsed twice with lysate, and added SDS-PAGE buffer.
We took the liquid from the above product for SDS-PAGE, as we can see, PhaP-HD5d5 combines the most products with PHA，whether it is electrospinning or nanoparticles.

Figure.8

Reference

[1] Xue Q, Liu XB, Lao YH, Wu LP, Wang D, Zuo ZQ, Chen JY, Hou J, Bei YY, Wu XF, Leong KW, Xiang H, Han J. Anti-infective biomaterials with surface-decorated tachyplesin I. Biomaterials. 2018 Sep;178:351-362. doi: 10.1016/j.biomaterials.2018.05.008. Epub 2018 May 9. PMID: 29778319.
[2] Wang, Cheng et al. “A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.” Antimicrobial agents and chemotherapy vol. 62,2 e01504-17. 25 Jan. 2018, doi:10.1128/AAC.01504-17

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]