Part:BBa_K1697000

SboA from B.subtilis 168

Subtilosin-A is a 32 AA antimicrobial peptide produced by Bacillus subtilis 168. This part contains only the Subtilosin-A coding region without its operons

Usage and Biology

Subtilosin-A is a polypeptide with a molecular weight of 3398.9, consisting of 32 usual amino acid and some non-amino acid residues. It has antimicrobial activity against a number of bacteria. It is a polypeptide that has antibiotic activity that coded and synthesized by microorganisms, called bacteriocin. Subtilosin A has classified as bacteriocin class V because this protein has unique circular structure rather than another class of bacteriocin. Circular structure of this protein caused by disulfide bond that formed in post-translation by modification protein.

characterization:

1) Sperm Motility Assay

Method: Sperm Motility Assay was performed to see the engineered subtilosin A effect on human sperm.

For motility assay, we use human spermatozoa. Ethical clearance was issued. Subject was informed and consented.

Sample was collected by self masturbation. It was immediately (within 1 hour) observed under light microscope. On counting sperm motility, we ask professional help from Departement of Biology Universitas Indonesia. The method was random, blind study. Modified Sutyak KE, et al was used to determine the sperm motility assay. 200μL of culture was mixed with 40μL of whole semen. The percentage of forward moving spermatozoa was documented.

Result:

Decrease in sperm motility was observed after intervention. We add Media + antibiotic as a control for observing sperm death due to addition of media +antibiotic (TB+tetracycline). C1, C2, and C3 was culture 1,2, and 3 respectively. WT represent wild type BL21 CP without plasmid.

File:Https://parts.igem.org/File:Tabel sboa sperm 123.PNG

File:Https://parts.igem.org/File:Tabel sboa sperm.PNG

Graph explanation: Y bar represent sperm motility, forward progressing sperm. X bar represent; Start – starting sperm motility without intervention; media+ antibiotic – sperm motility after addition of media and antibiotic; h0 – represent culture before induction, at the time of induction with IPTG; h-2 represent culture after 2 hours induction; and h-4 represent culture after 4 hours of induction.

Conclusion: Media containing antibiotic inhibit sperm motility by inhibiting its forward progression, thus, this data disrupt the other data. However, average of C1,C2, and C3 shows more sperm inhibition than wild type E. coli BL21 CP.

2) Well Diffusion Assay

Well diffusion assay was performed to see Subtilosin A effectivity as bacteriocin.

A 3 hour culture of M. luteus in Nutrient broth was spread in blood agar and Mueller Hinton Agar. The bactericidal activity was observed after incubation of 18 hours

Result:

There seem to be no clear zone in the blood agar or Mueller-Hinton Agar.

Conclusion: The bactericidal activity of Subtilosin A on M. luteus seems to be lost. This may be due to decreased biological activity of subtilosin on bactericidal capacity as the operon is not present (albA-albG)

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]