Part:BBa_K3351002

HBD3, an antimicrobial peptide.

Summary

The three human β-defensins, HBD1-3, are 33-47-residue, cationic antimicrobial proteins expressed by epithelial cells. All three proteins have broad spectrum antimicrobial activity, with HBD3 consistently being the most potent. Additionally, HBD3 has significant bactericidal activity against Gram-positive Staphylococcus aureus at physiological salt concentrations.Defensins are small, 3–5 kDa cationic proteins constrained by three disulfide bonds. As a class of proteins, they have broad microbicidal activity against Gram-positive and -negative bacteria, yeast, and some enveloped viruses, although specific defensin peptides often have defined spectra of activity. Like many other antimicrobial peptides, the defensin class of peptides is known to disrupt the membranes of microbes . It has recently been reported that in addition to their antimicrobial activity, defensins may act as chemokines, activating the adaptive immune response .HBD3 possesses bactericidal activity against Gram-positive and -negative bacteria, including multi-drug-resistant S. aureus, vancomycin-resistant Enterococcus faecium, and Burkholderia cepacia in addition to the yeast C. albicans .

Characterization

Figure.1 growth curve

Reference

[1] David J,Schibli,Howard N,Hunter,Vladimir,Aseyev,Timothy D,Starner,John M,Wiencek,Paul B,McCray,Brian F,Tack,Hans J,Vogel.The solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus.[J].The Journal of biological chemistry,2002,277(10):8279-89.
[2] Schroder, J. M. (1999) Cell. Mol. Life Sci. 56, 32–46
[3] Epand, R. M., and Vogel, H. J. (1999) Biochim. Biophys. Acta 1462, 11–28
[4] Harder, J., Bartels, J., Christophers, E., and Schroder, J. M. (2001) J. Biol. Chem. 276, 5707–5713
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]

BBa_K1033910 parameters

Burden Imposed by this Part:

Burden Value: 0.2 ± 2.4%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

CONTRIBUTION: UTokyo 2021

Authors iGEM UTokyo 2021, Rintaro Shimojo.

HBD3 has antimicrobial effects on yeast not only on bacteria, so we evaluate the effect of HBD3 on Saccharomyces cerevisiae.

Purpose
The UTokyo 2021 aimed to make HBD3 secreting S. cerevisiae for wound care. However, it is reported that HBD3 has antimicrobial activity against yeast [6], and secretion of HBD3 may affect the yeast growth of yeast. Therefore, the growth rate between the HBD3-secreting strain and the control strain had to be compared in our project.

Method
To evaluate the effect of HBD3 on S. cerevisiae, we evaluated the growth rate of HBD3-secreting yeast according to the following protocol. HBD3 secreting yeast was made by transferring (BBa_K3804013 BBa_K3804013)

1. Inoculate 200ul of saturated yeast culture into 3ml of YPD.
3. Incubate at 30°C and 200 rpm with shaking.
2. Measure the OD of the culture medium at regular intervals.

As a control, we use pRS316(which is a shuttle vector for yeast and E. coli) transferred into S. cerevisiae.

Results
As shown in Figure 2, HBD3 dramatically affected yeast growth during the exponential phase compared to control. This property was observed particularly at 250 minutes after the inoculation. However, since the final concentration was almost the same, HBD3 secretion was considered to have little effect on the number of S. cerevisiae in the end. Thus, HBD3 secretion yeast is expected to be sufficient for practical implementation. [[T--UTokyo--HBD3 Yeast.png]]

Figure 2: Effect of HBD3 expression on growth of S. cerevisiae. Orange:HBD3 expressing strain. Blue:control strain.