Designed by: LIN, WENXIN Group: iGEM18_TUST_China (2018-10-06)
φX174E
phiX174E is a kind of protein from the Bacteriophage phi-X174 which can induce the cell lysis.
The active machanism of this protein is that inhibits the cell translocase MraY activity that catalyzes the synthesis of lipid I, a necessary step for the host cell wall biosynthesis.
Usage and Biology
Gene E from the phiX174 phage encodes a protein that inhibits cell wall synthesis, ultimately leading to cell death and lysis (Bernhardt et al., 2000). Its function is to block the activity of MraY, an integral membrane enzyme responsible for a key step in bacterial cell wall biosynthesis (Chung et al., 2013). This protein acts independently and is sufficient to induce lysis, making it a simpler mechanism compared to the lysis processes of other bacteriophages, and easier to implement in bioengineered systems (Witte, 1990).
Group Information
Group: Princeton iGEM 2024 | Author: Camille Perez, Daniel Tu | Summary: We added a literature review along with our experimental data that characterizes the speed of the lysis gene and quantifies the amount of lysate released. We tested protein E under an inducible Tet promoter and found that 100 ng/µL of aTc was most effective for lysis. Though our sequences are the same length, there were several point mutations, which are accounted for in the part BBa_K5180925 (PhiX174_GeneE).
Images and Figures
Figure 1: PhiX174 protein E inhibits cell wall synthesis
This figure provides a simplified representation of how the phiX174 protein E indirectly causes cell wall breakdown by inhibiting the activity of MraY, a crucial enzyme in peptidoglycan synthesis. MraY catalyzes a key step in the formation of the bacterial cell wall by facilitating the transfer of peptidoglycan precursors. By blocking MraY’s function, protein E disrupts the synthesis of peptidoglycan, weakening the structural integrity of the cell wall and leading to cell lysis. This streamlined mechanism highlights the efficiency of protein E in bypassing more complex lysis processes used by other bacteriophages.
Figure 2: Effect of PhiX174 protein E on cell density
This figure presents the results of an experiment involving a plasmid containing the phiX174 gene E, placed under a Tet promoter, which was transformed into competent Stellar cells. Various concentrations of anhydrotetracycline (aTc) were added to induce the promoter, resulting in the transcription of gene E and the production of Protein E. Lysis of the bacteria occurred between hours 1 and 2, with the bacterial population remaining low until approximately hour 10. It is hypothesized that the population begins to recover after hour 10 due to the potential degradation of aTc. However, the significantly lower population of bacteria expressing Protein E, compared to the control, clearly demonstrates the effectiveness of Protein E in lysing the engineered bacteria. These results confirm that Protein E can successfully induce complete lysis in our system.
Figure 3: GFP released into supernatant when cell is lysed with phiX174 protein E
This figure presents the results of an experiment involving a plasmid containing the phiX174 gene E, placed under a Tet promoter, which was transformed into competent Stellar cells. Various concentrations of anhydrotetracycline (aTc) were added to induce the promoter, resulting in the transcription of gene E and the production of Protein E. Lysis of the bacteria occurred between hours 1 and 2, with the bacterial population remaining low until approximately hour 10. It is hypothesized that the population begins to recover after hour 10 due to the potential degradation of aTc. However, the significantly lower population of bacteria expressing Protein E, compared to the control, clearly demonstrates the effectiveness of Protein E in lysing the engineered bacteria. These results confirm that Protein E can successfully induce complete lysis in our system.
