Part:BBa_K3927018

mFA-HBD2

Coding sequence for the human immune protein, human beta defensin 2 fused to the mating factor alpha peptide for secretion in S.cerevisiae

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 85
  

Description

This part is a composite part comprising of part BBa_K792002 (Mating Factor Alpha) and part BBa_K3927017 (Human Beta Defensin 2).

Design

Due to problems associated with expressing recombinant lactoferrin in S. cerevisiae, an alternative antimicrobial peptide (AMPs) was required as a candidate biopesticide. After extensive research into literature and consulting with companies exploring the use of AMPs for agriculture use, Beta-defensins were chosen. Vaciome, a Singaporean synbio startup focusing on AMPs for livestock protection, agreed that in theory, defensins could function as robust antifungals. Jonathan Bester, CEO of Vaciome, strongly believed this project had strong potential to impact the future of crop protection and generously donated to a Human Beta Defensin 2 (HBD2) plasmid optimized for P.pastoris.

Characterisation

The HBD2 fragment was obtained by amplifying the HBD2 gene from the P. pastoris plasmid donated by Vaciome. Using Gibson Assembly, the HBD2 fragment was assembled into a plasmid with a Gal1 promoter upstream, with a 6xHis-tag fused to the C-terminus. The new construct was named pGmHBD2-H, and was transformed into BY4741 to test the expression, secretion, and functionality of HBD2 via galactose induction.

pGmHBD2-H was inoculated from glycerol stock in YPD-HygB and incubated overnight. Culture media was swapped to YPGR to initiate galactose induction. After 48 hours, the culture was spun down to isolate the media supernatant and the cell pellet. The media was concentrated by 100x and purified using centrifugal Ni-NTA columns. Cell pellet was washed and lysed, after which it was centrifuged, and the lysate supernatant was retained. SDS-Page was performed on the media, media concentrate, purified media, and media concentrate, as well as unpurified and His-tag purified cell lysates.

To test the functionality of the HBD2 produced, we performed a MIC assay using the agar diffusion method against E. coli to validate the HBD2 function with the MIC data from Vaciome. The total protein expression of HBD2 was subpar in comparison to the MIC data that Vaciome produced using HBD2 expressed from P. pastoris culture. A small zone of inhibition was produced only when we concentrated our media isolate by 100x, in contrast, Vaciome’s P. pastoris media isolate had enough HBD2 to produce a zone of inhibition when used neat. A possible explanation is that S. cerevisiae was not as efficient in expression and secretion of HBD2 as compared to P. pastoris.