<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

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.

Goal

To establish an inter-kingdom communication system between bacteria and human cells, we tried to integrate an existing signalling transmission systems. In many studies, human signal transmission systems have been discovered. Also, in iGEM community, quorum sensing is famous as a bacterial cell-to-cell communication.

One day, we found a previous publication that human cell can reveive AHL which is a signaling molecule used in quorum sensing. Based on the previous research, we decided to apply quorum sensing for the signal transmission from bacteria to human cells in our genetic circuit. However, according to a previous research, it's difficult that human cells synthesize AHL derived from bacteria. This is because AHL precursors need to be biosynthesized in human cells and to do so, we have to transduce nine kinds of genes (Bacteria II type fatty acid synthesis path) into human cells. Some studies show the attempts to make human cells synthesize AHL, but all the results are not successful.

For these reasons, we cannot introduce quorum sensing into bacterial co-culture system. In addition, after a series of transcriptome analysis, suitible compounds don't exist. In addition, compounds derived from human cells may influence not only bacteria but also other human cells. Thus, we decided to use plant hormones which are from neither bacteria nor human cells. According to some publications, this information processing mechanism unique to plants is also derived from bacteria and due to the similarity between them, the establishment of fusional signal transmission system is thought to be possible.

In this way, we established the following systems.

- Human cells and bacteria: Transcription control by integrating quorum sensing and NF-kB, transcription factor in mammalian cell
- Bacteria and plants: Transcription control by integrating signal transmission systems derived from bacteria and plants

We'll describe each mechanism.

Population of bacteria is a key point for a co-culture of bacteria and human cells. If bacteria overgrow, their inhabitance shrink and they end up extingishing. Therefore, we thought it's necessary to establish a system to sense the population of E. coli and respond to the change. To this end, we refered a previous research on the integration of quorum sensing and eukaryotic transcription control.

Step1: We transduced traI which codes C8 synthetase and as E. coli grow, C8 is synthesized and secreted. In human cells, the following two genes are transduced.

- traR which codes a receptor for C8 (one of signaling molecules in quorum sensing)
- relA-NLS-traR which includes transactivation domain of RelA in NF-kB

In the fusional protein, TraR works as a DNA-binding domain and a part of RelA works as a chimeric transcription factor.

Step2: After the complex receives C8, the chimeric transcription factor structurally changes and the active dimer is formed.

Step3: TraR in the active factor binds tra box located upstream of the target gene.

Step4: RelA TAD activates CMV minimal promoter and the expression of the downstream gene is induced.

In this way, we established fusional signal transmission system between bacteria and human cells. In addition to this system, human cells have to produce growth inhibition factors against bacteria. As described before, this signal must interact with not other human cells but only E. coli. Therefore, we decided to use one of plant hormones, cytokinin. This time, we chose iP (isopentenyl adenine from A. thaliana) One reason is the plant hormones don't affect the other mechanisms in human cell. The other is the signal transmission system of cytokinin is derived from TCS and the cytokinin can work as a signal for bacteria. Also, it was one of the important factors that we had to transduce only two genes for iP synthesis. In this system, two TCSs from E. coli and plant are integrated and iP works as a growth inhibition signal for E. coli.

TCS of E. coli

Step1: RcsC, which E. coli originally has, works as both receptor and His kinase and after it receives stimulus, self-phosphoration occurs.

Step2, 3, 4: Phosphoryl group transfers and eventually, the phosphoryl group reaches and binds RcsB, a responce regulator. A series of the reactions is called phosphrelay.

Step 5: Phosphorylated RcsB binds RcsA and a hetero-dimer is formed. The hetero-dimer binds transcription regulation region in cps operon and the downstream gene is expressed.

Fusional TCS

This time, as for reporter E. coli, we knocked out native RcsC and instead, we transduced ahk4 gene, which codes a iP receptor protein. In this genetic circuit, after AHK4 receives iP, Rcs TCS is activated. AHK4 is a identical protein to RcsC and according to previous studies, it can also work in E. coli.

Step1: After AHK4 receives iP, self-phosphorylation occurs.

Step2, 3, 4: Phosphoryl group transfers and eventually, the phosphoryl group reaches and binds RcsB, a responce regulator. A series of the reactions is called phosphrelay.

Step 5: Phosphorylated RcsB binds RcsA and a hetero-dimer is formed. The hetero-dimer binds transcription regulation region in cps operon and the downstream gene is expressed.

In this project, we transduced mazF gene which codes RNA interferase in sequences downstream of Cps promoter and designed a growth inhibition model for E. coli.

By using these two signal transmission systems, we established the following co-culture system.

Step1: C8, an autoinducer AHL, is produced and released from E. coli. With increased population of E. coli, the concentration of C8 increases in the culture medium of human cells (EA.hy926) and E. coli.

Step2: C8 binds with TraR expressed in EA.hy926 cells, and RelA-TraR-C8 complex promotes the expression of AtIPT4 and LOG1 by the specific signal pathway. The more AtIPT4 and LOG1 expression is promoted, the more Isopentenyladenine (iP) molecules are synthesized in EA.hy926.

Step3: EA.hy926-secreted iP binds with membrane receptor, AHK4, of E. coli.

Step4: iP activates AHK4 and His-Asp phosphorelay delivers phosphorylation signals . Eventually, it leads to activate CPS promoter and downstream mazF is expressed.

Step5 Cleavage of mRNA by a toxin, MazF stops the protein synthesis and the growth of E. coli.

To evaluate that this circuit actually works, we conducted the following assays.

Bacteria to human cells

- Transducing traI which codes C8 synthetase, observing the capability of C8 production and secretion and measuring the amount
- Transducing chimeric transcription factor (RelA/NLS/TraR) and inducive iP synthetase genes into human endothelial vascular cells (EA.hy926), and measuring the trascription level of the genes when C8 exist

Human cells to bacteria

- Measuring the activation level of fusional TCS after AHK4 on E. coli's membrane receive iP, a signal from human cells.

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