Part:BBa_K2500009

Protein E: Bacteria-Lysing Protein Encoded by Phage Phi X 174 with Engineered RBS

Protein E is a lysis protein originally encoded by the bacteriophage Phi X 147 and destabilizes the bacterial cell membrane resulting in cell death.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

CATE deploys protein E to release the cytotoxic agent within tumor cells upon heating the desired are to 45 °C with focused ultrasound. The exact mechanism of action of protein E has long been controversial and different models were proposed to explain its lytic function. It has been suggested that protein E activates a component of the E. coli autolytic system, that it inhibits cell wall synthesis in a manner similar to penicillin or that it oligomerizes into transmembrane tunnels. Both mechanisms would result in the release of cytoplasmic content and thus ultimately cell death.^{1 2 3}

However, it is now generally accepted that the most probable cellular target of protein E is an enzyme called translocase I encoded by the mraY gene. Translocase I is required for cell wall biosynthesis and its inhibition leads to cell lysis. The "transmembrane tunnel" model has largely been discarded and the tunnels are now attributed to faulty cell wall synthesis.¹

Design

Initially, we placed protein E downstream of the p_Lux promoter but due to the leakiness of the promoter no colonies could be obtained due to premature cell lysis. Therefore, we created a RBS library sing the Red Libs algorithm to find variants translating less protein E RNA in order to prevent premature lysis.

Characterization

We tested the function of heat-induced cell lysis by inducing E. coli TOP10 for 3 h at 45 °C in culture tubes in a shaking incubator. Because the fluorescence of each sample was different due to intrinsic noise, only the ratio of total fluorescence and fluorescence in the supernatant can give a cue about the lysis efficiency. Therefore we measured the total fluorescence and the supernatant fluorescence every hour for the induction period (Figure 1).

Figure 1: Protein E RBS library variant assay. Four library variants were selected, induced with a heat shock of 45 °C (bottom row) and the release of GFP (fluorescence) into medium was observed. Variant C exhibits the lowest leakiness of protein E at 37 °C. It is the engineered TlpA RBS and leads to a tight repression of protein E. The negative control consists of constitutively expressed GFP without protein E.

Variant 2 was selected as the optimal solution due to its superior translation initiation (TIR) rate.

Figure 2: Protein E RBS variants were sequenced and their calculated translation initiation rates compared.

Summary

• We rationally designed an RBS library in order to tune protein E expression strength.
• We managed to decrease expression levels at non-induced state far enough to successfully co-transform protein E with the thermosensing system.
• We showed that it is possible for our engineered bacteria to grow at 37 °C when transformed with the heat-inducible cell-lysis system.
• Upon induction at 45 °C, we showed that the cells lyse and release their protein-content into the environment.

References

1. W. Roof et al. "Engineering the perfect (bacterial) cancer therapy." Nature Reviews Cancer (2010): 785-794
2. W. Lubitz et al. "Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174." Journal of Bacteriology (1984): 385-387
3. A. Witte et al. "Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E." Journal of Bacteriology (1990): 4109-4114