Figure 1
Procedure and quality control of ghost production from E. coli O78:K80. The schematic representation of the E. coli O78:K80 bacterial ghost production procedure. A The pmET32c vector (Genebank: MN049497.1) contains the gentamicin-resistant, E lysis, and SNUC genes; the expression of the latter two genes is under the control of temperature-sensitive lambda-repressor CI857 (λ rep) and LacI inhibitors. The expression of these two genes is induced by increasing temperature from 28 to 42 °C and the addition of IPTG. Following the expression of E-lysis and SNUC (S nuclease), the E. coli O78:K80 is turned to the bacterial ghost which loses all cytoplasmic contents through the cell wall pores, and the DNA and RNA contents by enzymatic digestion. The other regulatory elements shown in the pmET32c vector include the LacI promoter (LacIpr), Lac operator (Lacop), and tac promoter (tacpr). The functionality of E-lysis and SNUC proteins in bacterial ghost production steps were checked and confirmed as shown in B–F. B Growth kinetics of E. coli O78:K80 harboring pmET32c vector was monitored by measuring the optical density of bacteria at 600 nm (OD600 nm) and colony number determination at different time points during the BG generation procedure. The OD600 nm reading shows a drop 60 min after E-lysis induction (28 °C to 42 °C) and increases as a result of the release of bacterial cell cytoplasmic contents. The time points are based on the minutes relative to E-lysis induction (0 min). C Scanning electron microscopy (SEM) of E. coli O78:K80 ghosts before (left panel) and at the end of washing steps (right panel). Black arrows in the right panel and inset indicate the lysis-induced pores in BGs and red arrows in the left panel show the release of cytoplasmic contents through these pores. D The expression of SNUC results in the degradation of nucleic acids. The total DNA content at different time points of BG production steps within pellets (left panel) and the supernatants (right panel) were visualized by 0.7% agarose gels electrophoresis. E, F Quantitative analysis of pmET32c vector content of BGs before and after 3 rounds of washing via absolute quantitative real-time PCR based on the measurement of copy number. The amplification and standard curves of pmET32c vector copy numbers are shown. It is illustrated that at the end of the BG production process and after the first and third washings of E. coli O78:K80 ghosts, the vector copy numbers fell out of detection limits below 100 copies. No washing: green triangle; After first washing: red square; and after third washing: blue dots.