Project Description

Background

How can we define a human organism? Is it simply a group of human cells? It's said that in our body, there exist not only 3.0*10^13 human cells but also 3.8*10^13 bacteria. That means the mass of bacteria reaches 0.2 kg. Bacteria and human have co-existed for a long time, for instance, intestinal flora and oral flora and it's obvious that bacteria play an important role in our body. As a way to improve one's intestinal environment, recently, a new therapy that transplants intestinal flora in healthy body, has been developed. This example shows that if we can intentionally make specific strains of bacteria stay in our body, it may be possible to change our characteristics.

To sum up, we'd like to define a human organism as "an organism in which human cells and bacteria co-exist." In iGEM community, it's been a standard to use single organism in project and it's not an overstatement that most teams don't take it into account that in a real world, multiple kinds of organisms co-exist and the ecosystem is sustained by their mutual dependence. Therefore, to target "true human organism", it's necessary to establish the system that human cells and bacteria co-exist under in vitroconditions.

In previous iGEM projects, two teams from 2011 and 2014 competitions tried to co-culture human cells and bacteria. However, the former team couldn't obtain persuasive data and Team ETH Zurich in 2014 set the goal as a short-term co-culture, which means that they didn't establish a system that co-cultures them for a long term.

As long as we can assume, there are the following reasons that interfere the establishment of co-culture system.

- A growth rate of bacteria surpasses that of human cells.
- Few studies show signal exchange mechanism between them.
- Bacteria are usually excluded by human immune system.

If we can establish a co-culture system, we can find a way to achieve population balance to sustain the co-existence and apply for a medical field like a cancer treatment. In addition, since human and bacteria have originally co-existed, the establishment of a co-culture system will contribute to the development of organism closer to life.

Concept and Mechanism

Concept

Our original goals are as follows:

1) Establishing an artificial inter-kingdom communication system between human cells and bacteria.
2) Creating a co-culture model using the inter-kingdom communication and designing ‘Coli Sapiens,’ a new type of human strengthened by bacteria

To achieve the first goal, we needed a new cell-to-cell communication system because native and direct communication systems between human cells and bacteria were little known. Thus, we decided to integrate signal transmission system among three kingdoms.

To achieve the second goal, we chose the essential parts in a complex co-culture system between bacteria and human cells. The reason why co-existence between them has not been developed under in vitro conditions is that a growth rate of bacteria surpasses that of human cells. Thus, when we designed the mathematical model, we emphasized a population of bacteria as one of the biggest factors to establish a co-culture system.

Mechanism

Results

traI Improvement Assay

At an early stage of our project, we simulated the whole co-culture system using parameters from the C8 production rate of E. coli, the iP production rate of human cells and growth inhibition rate of mazF. The simulation showed that the C8 production rate is not enough to induce the iP production and as a result, E. coli overgrow.

To increase the C8 production rate, we improved the previous genetic circuits in two ways.

- - Introducing various point mutations into CDS of the traI gene and finding a strain whose C8 production rate increases
- - Adding SAM (one of the C8 materials) to culture medium and promoting the C8 production

As a result of the improvement, the concentration of C8 which E. coli produce increased by about 100 folds and it has been possible to induce iP synthesis in human cells from an early stage of E. coli's growth.

Chimeric Transcription Factor Assay

As for human cells' constructs, we synthesized chimeric transcription factor and iP synthetase genes. In the assay, first, we transduced the constructs. Then, we cultured the cells in which the constructs are successfully transduced and added C8 from E. coli. After the addition, we checked the transcription of atipt4 and log1 (part of iP synthetase genes) using transcriptome analysis. From this result, we concluded that human cells received C8 from bacteria and successfully produced iP.

AHK4 Assay

We transduced ahk4 into E. coli (KMI002 strain) and cultured them. Then, we added iP and after AHK4 received iP, cps promoter was activated and downstream lacZ is expressed. (lacZ expression was confirmed by blue-white screening.) In conclusion, it turned out that AHK4 can receive iP and induce the gene expression of the downstream genes, which means in a larger scale, E. coli can receive growth inhibition factors from human cells and inhibit the own growth.

Simulation

We simulated the whole co-culture system again using the assay data. The simulation result showed human cells can control the population of E. coli and the population oscillates.

Team:TokyoTech/Description