Bombolitin Vector Design

We sub-cloned the bombolitin mRNA sequence from the venom of the bumblebee Bombus ignitus to pGEX-61 by process of DNA synthesis. From this, we found out that bombolitin has a 2kDa mature peptide with approximately 18-amino acid residues. Since we were concerned with the stability and activity of the peptide, we decided to modify the peptide by adding 3 amino acids which were phenylalanine, leucine, and tryptophan at the C-terminal to test the stability and activity. We then had to transfer these inserts separately to PET32a for protein expression.

DNA Amplification of Bombolitin

We successfully amplified Bombolitin using PCR and checked the results that included the primer using electrophoresis.

DNA Purification of Bombolitin

We used the process of PCR purification to achieve the purified DNA of bombolitin. Above is the result of the gel electrophoresis that we performed to check the results of the purified DNA.

Ligation of Bombolitin with pET23-a

From ligating the bombolitin with pET32a, we successfully modified the c-terminal with FFF, LLL, and WWW respectively. After the process of ligation and cut by BamHI and SalI, the results showed that pET32a-Bom LLL succeeded for insertion to pET32a.

Protein Expression of pET32a-Bombolitin

From the protein expression of pET32a-Bombolitin, the results showed that the rBombolitin was expressed after induced by 0.5 mM IPTG at 37 degrees celsius for 4 hours.

Protein Characterization of pET32a-Bombolitin

Through the process of protein characterization, the results showed that the rBombolitin 22.04 kDa bound to the Ni-NTA column was eluded by 300mM imidazole. We also tested using agar diffusion assay and the result showed that 500 µg of the modified Bombolitin peptide displayed significant activity against Ralstonia solanacearum.

The negative control was buffered saline, which shows no antimicrobial activity. 500ug of original Bombolitin also shows inhibition of Ralstonia, but not as significant as the modified Bombolitin or positive control. For the modified Bombolitin, the diameter of inhibition is 5mm. The positive control used ampicillin which has an inhibition of 10mm. We have concluded that the minimum inhibitory concentration of modified Bombolitin is 500ug.

Infect into E.coli TG1 (First round)

Infect into E.coli HB2151 (Second Round)

The graph above shows the results from the first and second round of biopanning, each labeled respectively. In the first round of biopanning, we put the nanobodies at a concentration of 10e11 infectious particles within the sample and received an output of 10e4 infectious particles within the sample when we infected E.coli TG1. We then precipitated the phage. In the second biopanning round, we continued to take the phage that we precipitated by putting it at the same concentration of 10e11. As a result, we got a higher output of 10e8 infectious particles within the sample after infecting it into E.coli HB2151, indicating that we got more specific nanobodies.

During the first round of biopanning conducted, E.coli TG1 was infected in order to obtain a colony, then inoculated at a large scale of 10 mL, and finally spread. For the second round of biopanning, the bacteria was infected into E.coli HB2151 so that the codon became readable.

Furthermore, Indirect Enzyme-Linked Immunosorbent Assay (ELISA) was used to test which nanobody binds best with the R.solanacearumout of the 20 PBS clones. The clones were compared with PBS in order to identify the most specific nanobody. Based on the data graph above, clones number 1, 7, 8, and 17 will be able to bind best with R.solanacearum . This is because they were the most distinct when compared to the PBS control.