Team:UNICAMP-Brazil/Coliguard/Recognition

The Coliguard - Recognition

Introduction

We aim to create bacteria that can protect themselves against contaminants. So, our bacteria need to be able to kill other organisms which may appear in the culture medium.

• Why not create a continuous mechanism of eliminating contaminants?

A simple way to do this would be to create bacteria that express a toxin constitutively. However, if the bacteria produce the toxin continuously, they will compromise their metabolic energy for the production of the toxic compound and, in addition, the production of large amounts of these antibiotic substances may contribute to the problem of antibiotic resistance. Moreover, the toxin itself would be a type of contaminant in the industrial process, requiring resources to be allocated in order to remove the toxin from the medium and purify the compound of interest.

• Therefore, we need a contaminant-sensitive recognition system!

We need to create a contaminant-sensitive mechanism that will allow the detection of the contaminants and trigger the killing mechanism. In this case, the toxin will be produced only when the contaminants are present.

• How will our bacterial guards recognize the contaminants?

Our idea is based on the premise that the engineered E. coli must be able to recognize contaminants in the culture medium as non-self. Thus, our bacteria need to recognize a specific signal that they don’t produce, but which are broadly produced or presented by potential contaminants. Thinking about the potential signals, we found two candidates: the AI2 (auto inducer 2) and the conjugation recognition mechanism.

• Using AI-2 as a signal. How?

As most bacterial species produces AI2 as a secondary metabolite, we decided to use this compound as a recognition factor. Our E. coli will be an AI2- strain and won´t produce native AI2. The presence of AI2 in the culture medium will then indicate the presence of contaminants, which will be recognized by an AI2 sensitive promoter present in our E. coli. This promoter will control the differentiation and killing mechanisms (see the links for more information).

• But, what if the contaminants do not produce AI-2? In these situations, our E.coli will use another recognition mechanism, based in conjugation. In conjugation, a donor bacterium only conjugates with organisms that don’t have the same conjugative plasmid. Bacteria that already have the plasmid display membrane proteins that prevent the conjugation with other bacteria that have the same conjugative plasmid. (reference) Therefore, if our E.coli has a conjugative plasmid, they will only conjugate with different organisms, which will be the contaminants.

Nevertheless, conjugation is a process that does not occur between E. coli and any other organism. There is a type of specificity to this process. A possible alternative to reduce this specificity is using multiple replication origins in the same conjugative plasmid (see Recognition by conjugation – lab strategy and results). However, this solution fails to address the whole problem.

We think that a better way to improve the recognition system is to combine the conjugation signal with the AI-2 recognition mechanism.

Our recognition system: a combination of conjugation and the AI-2 recognition mechanism!

To improve our recognition system, we will use a signal produced at the beginning of the conjugation process in order to stimulate the production of AI-2 in our E.coli. This signal will induce the promoter pY, which controls the expression of the conjugation-related genes. This promoter will then control the expression of the AI-2 gene in our E.coli. As a result, when conjugation begins the promoter will be induced and thus cause an up-regulation in AI-2 gene expression. In this way, the conjugation process will activate the killing and differentiation mechanisms in a way that is AI-2-dependent.

Combining these two recognition systems, our E. coli will be able to recognize a vast group of contaminants.