The Coliguard: Project Overview

• In our Coliguard System, 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 in the ability of the engineered E. coli be able to recognize contaminants in the culture medium. Thus, our bacteria need to recognize a specific signal that they don’t produce, but 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.

• 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 impaired in the synthesis of this compound, the synthesis will only happens when the cells receive the conjugation signal. 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.

• 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. Therefore, if our E.coli has a conjugative plasmid, they will only conjugate with different organisms, which will be the contaminants.

• For the conjugation, 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.

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

• After the signal indicating the presence of a contaminant, our Killer cell has three ways to kill its target: It can be by the Hemolysin Secretion System, Kamikaze System or Colicin System.

• The Hemolysin Secretion System core is a biobrick with the hlyB and hlyD and the hlyA signal peptide. hlyB and hlyD genes codify for the proteins that constitute the transporter that is able to transport outside the cell proteins with the hlyA signal peptide. The lambda phage lysozyme fused to this signal will be transported outside the cell and will attack the Gram Positive contaminants lysing them.

• The Kamikaze System induces a strong expression of the T4 endolysin when the Killer receives the contaminant signal. The endolysin is accumulated inside the cell and when its concentration reaches a threshold the own Killer is target of the endolysin breaking the cell wall and lysing the cell, consequently the endolysin is free in the medium to attack the Gram Positives contaminant.

• The Colicin System is our most accurate system. After the conjugation between the Killer and the contaminant, the contaminant receives a plasmid with the colicin gene. This gene is expressed producing the E2 colicin which act as an endonuclease cleaving the contaminant’s DNA leading it to death. The Killer cell have a immunity gene capable of blocking the colicin activity.