Part:BBa_K4990015

HokB time bomb

To ensure patient safety, a time bomb based on a weak RBS was designed to prevent excessive proliferation of engineering bacteria. Once the engineering bacteria has permeated the tumor microenvironment, the expression of toxin is automatically activated. The accumulation of toxin is slow due to the effect of the weak RBS, thereby preventing immediate death of the engineering bacteria. During this time frame, it is possible to engineer bacteria that can release DEH we design, and successfully accomplishing the mission of dual killing. As time passes, the accumulation of toxin will eventually reach a certain threshold, at which point the engineered bacteria will be killed under their own action, thereby completing the suicide process in vivo.

I. Toxin

As HokB belongs to the Hok/Gef family, it is predicted to be a single-span membrane peptide，which is a single-span membrane peptide with a long, negatively charged C-terminal periplasmic tail and a short, positively charged N terminus extending in the cytoplasm. HokB has the ability to selectively insert into the cellular membrane and aggregate to create pores with a diameter of around 0.64nm, which alters the membrane potential, depolarize the membrane, hinder ATP synthesis, decrease the cell's energy ratio, and lead to ATP leakage, ultimately resulting in cell death.

II. Weak RBS

We incorporated a weak RBS (BBa_B0033) upstream of the gene encoding the toxin and downstream of the hypoxia-responsed promoter. Once situated in the TME, bacterial transcription of the toxin commences in response to hypoxic stimuli. Subsequently, due to the presence of the weak RBS, the translation and accumulation of the toxin occurs at a subdued pace. The engineered bacteria will carry out dual killing within the specified time frame and undergo degradation once the threshold of toxic protein accumulation is reached. This will result in self-destruction, preventing excessive propagation of the engineered bacteria in vivo.

III. TA System Library

In the development of the suicide switch in the CPU-CHINA 2023 project, we utilised various components from the TA system and discovered its versatile applications beyond this scope. Therefore, we have created a library for the TA system and provided a concise introduction to it, which briefly arranged and condensed its composition, types, targets, present application scenarios, and techniques to benefit future teams in utilizing these components.

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]