Team:UNICAMP-Brazil/Project

The Microguards: Overview

Nowadays, there are numerous industrial processes that use microorganisms such as Escherichia coli and Saccharomyces cerevisiae to produce compounds of interest, like insulin, ethanol and various enzymes. The success of these processes depends on the absence of contamination by other microorganisms in the culture medium. The presence of contaminants in a fermentation process reduces its efficiency due to competition between the contaminant and the fermentative organism, causing losses of 5 to 10% of the gross production. To try to solve this problem, the aim of our project is to engineer strains of E. coli and S. cerevisiae that are able to recognize and destroy contaminants during industrial processes.

The Coliguard: Our Model

We want our engineered E. coli to be able to recognize and destroy contaminants in culture medium. Since we want our bacteria to show maximum efficiency during the industrial process, we decided to create two different lineages of E. coli: the worker lineage – responsible for executing the industrial process – and the killer lineage – responsible for detecting and killing the contaminants. Both lineages are going to be simultaneously present in the culture medium, but their proportion will vary due to the presence or absence of contaminants. In the absence of contaminants, the amount of worker cells will be much higher than the number of killer ones, so that the industrial process will occur at its maximum efficiency. In the presence of contaminants, killer cells will induce surrounding worker cells to differentiate into more killer cells in a transient manner. After elimination of contaminants by the killer lineage, the proportion of worker and killer cells will return to its original rate.

Based on our model, we divided the project into three subparts.

1. Recognition mechanism
2. Differentiation mechanism
3. Killing mechanism

1) Recognition mechanism

Our idea is based on the premise that the engineered E. coli most be able to recognize contaminants in the culture medium as non-self. As most bacterial species produces AI2 (auto inducer 2) 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 indicates the presence of contaminants, which will be recognized by an AI2 sensitive promoter present in our E. coli.

In the absence of contaminants, the amount of worker cells will be much higher than the number of killer ones, so the industrial process will occur at maximum efficiency.

Contaminants in the culture medium are recognized by the presence of AI2.

2) Differentiation mechanism

The initial differentiation mechanism is based on a random slippage mechanism that will determine the expression of a CRE recombinase in a small percentage of cells. This device is an adaptation from the device presented by the Caltech 2008 iGEM team. When expressed, the CRE recombinase will remove a device from the genome – containing a gene involved in the cell cycle and a gene that represses conjugation – and thus lead to the differentiation into killer cells. Killer cells are unable to reproduce, but able to conjugate. This device is an adaptation of the device presented by the Paris 2007 iGEM team. Moreover, the presence of AI2 in the culture medium will also trigger the expression of CRE recombinase and thus induce the differentiation of more worker cells into the killer cells, so the proportion of killer cells will be elevated during the decontamination process. After a certain number of generations, the proportion of killer and worker cells will return to its original state, due to the killer cells being unable to reproduce.

3) Killing mechanism

We decided to use conjugation as a sensor that will trigger the killing mechanisms. Our E. coli is going to be an F+ strain, carrying a modified version of the conjugative plasmid pPed100 containing a killing gene. Only the killer lineage will be able to conjugate because in the worker lineage the pPed100 plasmid will be repressed. Since most bacterial strains in nature are F- our F+ E. coli will be able to conjugate with most contaminants. There will be two killing mechanisms, one carried by the modified pPed100 plasmid into the contaminant and another triggered by the conjugation signal. This secondary mechanism is necessary to stall contaminant growth because conjugation may take some time to occur and may be impaired due to culture conditions. This secondary killing mechanism will be triggered by the presence of a diffusible conjugation signal and will be able to induce neighboring killer cells, even when not conjugating. Such diffusible signal and corresponding promoter have never been described, but we’ve found promising candidates and their characterization is the main challenge for this project.

The Yeastguard

Industrial ethanol production occurs in open vats with over 500,000 L. This process is mostly hindered by lactobacilli contamination, which produces lactic acid as their main metabolic product. Once contaminated, the whole vat must be discarded and cleaned, thus generating losses ranking in the millions of dollars.

We want our engineered yeast to be able to detect and control the proliferation of lactobacilli by introduction of a simple genetic device. The device must be able to recognize the presence of lactate in the medium during fermentation. In order to allow the entrance of lactate in the cell, we will construct a gene coding for a lactate transporter under the control of a constitutive promoter. Once in the cell, this metabolite will induce the transcription of the gene coding for killer enzyme, lysozyme, which is the most widely used antibiotic for lactobacilli decontamination.

The greatest challenge of this project consists of characterizing a lactate-inducible promoter that is not subjected to glucose catabolic repression, since the device must be activated during the process of sugar fermentation of the diauxic shift of S. cerevisiae.