After brain storming, we decided to use Escherichia coli colonies to simulate human community to show the Relationship Schema between groups. In order to create corresponding relationship between colonies, we designed two opposite mechanisms – “cooperative” and “aggressive” as follows:

Ideas

Cooperation is the process which organism groups working or acting together for common, mutual, or some fundamental benefit, as opposed to working in competition for selfish benefit (1). Thus, the goal of degrading cellulose into glucose for living was set as their common benefit.

Overview of cooperative

In our design, cellulose was set as the sole carbon source in the medium, and E. coli cannot use cellulose directly without any exogenous gene; the ONLY way they can survive is to produce cellulases to degrade cellulose into glucose for living.

To select suitable cellulases, we found different classes of enzymes involved in cellulose degradation by bacteria. The degradation of cellulose undergoes three stages with different enzymes (Fig. 1) (2). Endoglucanase A (Abbr. CenA, gene from Cellulomonas fimi), exoglucanase (Abbr. Cex, gene from Cellulomonas fimi) and beta-glucosidase (Abbr. Bgl1A, gene from Saccharophagus degradans) were finally selected to degrade cellulose through collaborative cooperation.

Fig. 1. The schematic of cellulose degradation by CenA, Cex and Bgl1A.

From Cellulose to Cellobiose

In order to achieve cooperation between two groups of E. coli, CenA and Bgl1A were selected and expressed in one group of the E. coli, while Cex and Bgl1A in the other. As cellulose cannot be transported into cells, and enzymes selected cannot be released to extracellular spontaneously, the enzymes should be secreted into the medium to degrade cellulose to achieve cooperation by certain means.

Two different secretion mechanisms of enzymes were designed: YebF-cellulase fusion protein and Kil secretion cassette.

YebF fused cellulase:

YebF, a secretion protein of E. coli (3, 4), was fused with N-terminal of cellulases through its C-terminal, which could be secreted together (Fig. 2, cellulases with protein YebF fused).

Fig. 2. The schematic of secretion due to protein YebF fused with cellulases

However, it is worth mentioning that the secretion mechanism of YebF is still not clear (3). We just engineered it to achieve the purpose of proteins secretion in our project.

Kil secretion cassette:

Protein Kil, a kind of bacteriocin-release protein (BRP) (5), was used to permeabilize the outer membrane and then to secrete protein inside of cells into the culture medium (Fig. 3).

Fig. 3 The schematic of Kil secretion cassette.

High level induction of the kil gene results in cell lysis and death (6), so promoters with different intensities (BBa_J23112, BBa_J23109 and BBa_J23114, Fig. 4) were used to regulate the expression of kil gene. The enzymatic activity of protein secreted into the culture medium was subsequently assayed to select the most suitable promoter.

Fig. 4. Gene circuits initiated by BBa_J23112/109/114 in the Kil secretion cassette section.

From Cellobiose to Glucose

After cellulose in culture medium was degraded into cellobiose by CenA and Cex, both of the two groups of E. coli could use cellobiose as substrate since they had exogenous gene bgl1A. However, cellobiose still needed to be transported into cell for usage.

We found that cellobiose can be transported into cells by the beta-galactoside permease (Fig. 5) encoded by the lacY gene in the lac operon (7), which could be achieved by induction of lactose or its analogues.

Fig. 5. Cellobiose could be transported into cell due to the function of beta-galactoside permease coded by lacY.

Once cellobiose is transported into cell, beta-glucosidase can function at the final step of cellulose degradation, and glucose is generated for living finally.

Fig. 6. The complete schematic of "cooperative" in our project.

Thus, the cellulose will be degraded due to the cooperation of two groups of E. coli, and they both survive (Fig. 6). In summary, according to the thoughts we depicted above, combining the results we obtained, we constructed the gene circuits (Fig. 7) to achieve the cooperation.

Fig. 7. The gene circuits to achieve cooperation between two groups of E. coli. (A) The gene circuits (BBa_K2922021) was constructed according to the function of Kil, CenA and Bgl1A in one group of E. coli. (B) The gene circuits (BBa_K2922022) was constructed according to the function of Kil, Cex and Bgl1A in the other group of E. coli.

Background

As a kind of bacteriocin secreted by Escherichia coli, colicin can kill other related bacteria that cannot secret specific immunity proteins, which is believed to regulate the number of bacteria (8).

In our design, two types of colicins were used to endow two groups of E.coli the ability to attack each other, thus presenting an aggressive scenario under certain induction conditions (Fig. 8).

Colicin-E1, Colicin-N and the corresponding immunity proteins and lysis proteins selected will be presented in the following details.

Fig. 8. Two strains of E.coli BL21 (DE3) attack each other by using Colicin-E1 and Colicin-N.

Colicin-E1 kit:

Colicin-E1: Encoded by gene cea, Colicin-E1 is a type of pore-forming colicin. This class of transmembrane toxins depolarize the cytoplasmic membrane, leading to dissipation of cellular energy (8).

Eimm：Abbreviated as Eimm, the molecular weight of Colicin-E1 immunity protein is 13 kDa, and the protein is encoded by gene imm. In wild strains of E.coli, the protein is able to protect cells, which harbors the plasmid ColE1 encoding Colicin-E1, against Colicin-E1. Immunity proteins interact with the pore-forming domain in the inner membrane. And the inactivation of Colicin-E1 occurs via interactions between the voltage-gated region and the transmembrane helices of the immunity protein. This protein will prevent E.coli secreting Colicin-E1 from killing themselves (10).

Ekil: Abbreviated as Ekil, lysis protein for Colicin-E1 is a 4.8 kDa protein, which is encoded by gene kil. The lysis protein is required for both Colicin-E1 release and partial cell lysis (8).

Fig. 9. The schematic of E.coli BL21 (DE3) killing other bacteria by using Colicin-E1 kit.

Colicin-N kit:

Colicin-N: Similar to Colicin-E1, Colicin-N is also a type of pore-forming colicin, encoded by gene cna. This class of transmembrane toxins depolarize the cytoplasmic membrane, leading to dissipation of cellular energy (8).

Nimm: Abbreviated as Nimm, the molecular weight of Colicin-N immunity protein is 15 kDa protein, and the protein is encoded by gene cni. In wild strains of E.coli, the protein is able to protect cells, which harbors the plasmid ColN encoding Colicin-N, against Colicin-N (9). This protein will prevent E.coli secreting Colicin-N from killing themselves (8).

Nkil: Abbreviated as Nkil, lysis protein for Colicin-N is a 5.6 kDa protein, which is encoded by gene cnl. The lysis protein is required for both Colicin-E1 release and partial cell lysis (9).

Fig. 10. The schematic of E.coli BL21 (DE3) killing other bacteria by using colicin-N kit.

In present work, both of Colicin-N and Colicin-E1 could be secreted into extracellular medium successfully and were able to kill other groups. In order to simulate specific situations, these two colicins were expressed through different inducers to simulate unidirectional or bidirectional aggressive relationships. Arabinose promoter and T7 promoter were selected as the promoter for transcription of Colicin-E1 kit and Colicin-N kit respectively (Fig. 11). When lactose (or its analogues) or arabinose were added to the culture environment, the relative promoter was induced to initiate transcription of these proteins, which means the aggressive behaviors would be executed (Fig. 8).

Fig. 11. Genetic circuits of Colicin-E1 kit and Colicin-N kit. (A) The gene circuit of Colicin-E1 kit: BBa_K2922039. (B) The gene circuit of Colicin-N kit: BBa_K2922038.

Reference

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