Description
With the rise of multidrug resistant bacteria, hospital patients are in increasing peril from common pathogens normally treated with antibiotics as treatment options become depleted. Out of all MDRs, Stenotrophomonas maltophilia, a Gram negative, aerobic bacilli, is of particular concern as it is highly pathogenic and resistant to many antibiotics. In particular danger of S. maltophilia infections are those with compromised immune systems such as cystic fibrosis (CF) patients. In CF patients, following treatment, biodiversity of anaerobes is generally reduced however, persistent strains of P. aeruginosa and S. maltophilia resistant to antibiotics linger causing further complications. Their resistance to multiple antibiotics makes developing alternative or synergic treatments gravely urgent. While phage cocktail therapy has proven promising, few phages are well characterized yet as an antimicrobial therapeutic for S. maltophilia. Tailocins, a type of bacteriocin, are antimicrobial polypeptides first characterized in 1954 and are returning to great interest as a promising alternative to antibiotics and phage therapy. Their killing simplicity, stability, and solubility give them great potential. Despite the potential antimicrobial applications of tailocins, little is known of their assembly properties as most current studies characterize novel tailocins. Insight into assembly will facilitate engineering projects such as retargeting tailocins to kill other bacterial species as many tailocins are narrow spectrum. In this study, we investigated the assembly properties of maltocin P28 derived from strain CF409 of S. maltophilia by cloning each individual structural gene, transforming into and expressing each gene individually in E. coli, and developing methods of preparation for transmission electron microscopy. We managed to successfully clone 11 out of 14 of the maltocin structural genes, verified the sequence in each clone using Sanger sequencing and colony PCR, and developed methods for freeze-thaw lysis, protein purification and storage, and solubility tests. We also assessed clone growing trends at various concentrations of IPTG and show that 1 mM IPTG induction at 30°C has no negative effects for most clones.
