Results
Screening for antimicrobial peptide producing thermophilic bacteria
The first step in our project was to select the best strain capable of producing antimicrobial peptides. To do this, we used the spot-on-lawn assay to screen 39 thermophilic isolates that had been previously isolated from a Saudi Arabian hot spring for their capacity to generate AMP. We used proteinase K to treat the thermophiles to verify that the antimicrobial agents they produced were proteinaceous. Following treatment with proteinase K, antibacterial activity disappeared, demonstrating the proteinaceous nature of the antimicrobial agent produced.
Table 1: Thermophilic bacteria screened for their ability to produce antimicrobial peptides
| S/N | Thermophilic bacterial Strain | Diameter of inhibition (mm) | Diameter of inhibition after treatment with proteinase K |
|---|---|---|---|
| 1 | 2 | 18 | 2 |
| 2 | 3 | 16 | 0 |
| 3 | 4 | - | - |
| 4 | 5 | - | - |
| 5 | 6 | 17 | 1 |
| 6 | 7 | 17 | 2 |
| 7 | 9 | 20 | 0 |
| 8 | 10 | - | - |
| 9 | 11 | 15 | 0 |
| 10 | 12 | 13 | 2 |
| 11 | 13 | 24 | 0 |
| 12 | 14 | 23 | 2 |
| 13 | 16 | - | - |
| 14 | 17 | 13 | 0 |
| 15 | 18 | 18 | 2 |
| 16 | 20 | - | - |
| 17 | 22 | 26 | 5 |
| 18 | 23 | - | - |
| 19 | 30 | 20 | 3 |
| 20 | 31 | 10 | - |
| 21 | 34 | 20 | 0 |
| 22 | 35 | - | - |
| 23 | 37 | - | - |
| 24 | 38 | 15 | 2 |
| 25 | 40 | - | - |
| 26 | 41 | 13 | 2 |
| 27 | 42 | - | - |
| 28 | 43 | 15 | 0 |
| 29 | 45 | 12 | - |
| 30 | 47 | - | - |
| 31 | 49 | - | - |
| 32 | 51 | - | - |
| 33 | 52 | 15 | 5 |
| 34 | 54 | 15 | 0 |
| 35 | 55 | 20 | 6 |
| 36 | 56 | 20 | 1 |
| 37 | 57 | - | - |
| 38 | 58 | - | - |
| 39 | 59 | 22 | 0 |
Considering that strain 13 produced the largest inhibition zone, which disappeared after treatment with proteinase K, we choose strain 13 (Brevibacillus borstelensis AK1) for further analysis.
Figure 1: Antimicrobial activity of Brevibacillus borstelensis AK1 against Staphylococcus aureus (a) Without proteinase K treatment. (b) with proteinase K
Identification of antimicrobial peptide genes of Brevibacillus borstelensis AK1
Using NCBI blast., Interpro, SignalIP, and antiSMASH, we examined the entire genome sequence to identify new antimicrobial peptides expressed in B. borstelensis AK1.
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&DATABASE=n/a&QUERY=&SUBJECTS=Six putative antimicrobial peptide genes were found using a sequence alignment-based method (for further information, see the bioinformatics section).
Result after sequence alighnment
| 1 | lcl|ORF278_APBN01000001.1:15207:15323 |
| 2 | lcl|ORF316_APBN01000001.1:29763:29900 |
| 3 | lcl|ORF330_APBN01000001.1:37251:37376 |
| 4 | lcl|ORF393_APBN01000001.1:28499:28422 |
| 5 | lcl|ORF396_APBN01000001.1:27719:27651 |
| 6 | lcl|ORF587_APBN01000001.1:41121:41053 |
The presence of signal peptides from the predicted gene was verified by the bioinformatics tools Interpro and SignalIP. Results indicated that all the predicted peptides were destined for release beyond the cell membrane, as evidenced by the presence of signal peptides.
Biosynthesis and Purification of predicted AMPs
The genes we aimed to assemble into a backbone were all synthesized by twist bioscience and IDT. The genes were assembled using Type IIS, although the correct band length could not be obtained. Furthermore, no growth was produced by transformation once our competent cells were prepared. Please refer to our experiment page for the lab procedures we used.
We are currently working on improving on our construct. Further results will be presented on our judging section.