What are we doing?

Our original goals are as follows:

• Establishing an artificial inter-kingdom communication system between human cells and bacteria.
• 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 ３つのkingdomのシグナル伝達システム.

As a result of brainstorming in the team, we established the following systems

• traI and chimeric transcripntion factor
  • Transcription control by integrating quorum sensing (bacterial cell-to-cell communication) and NF-kB, transcription factor in mammalian cell. We used this system the signal transmission from bacteria to human cells.
• Bacteria and plants
  • Transcription control by integrating signal transmission systems derived from bacteria and plants. We used this system the signal transmission from human cells to bacteria.

The details are described below.

Bacteria to Human cells

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 traIwhich 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.

Next, 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.
• Step5: 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.
• Step5: 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.

To evaluate that these systems actually works, we conducted the following assays.

• Bacteria to human cells
  • traI Assay and traI Improvement Assay
  • Transducing traI which codes C8 synthetase, observing the capability of C8 production and secretion and measuring the amount
  
  
  • Chimeric Transcription Factor Assay
  • 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
  • AHK4 Assay
  • Measuring the activation level of fusional TCS after AHK4 on E. coli's membrane receive iP, a signal from human cells

To achieve the second 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 ３つのkingdomのシグナル伝達システム.

To achieve the second goal (creating a co-culture model and designing a new type of human strengthened by bacteria), 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 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.