Project Description

Contents

i. Introduction

ii. Goal and Approach

iii. Mechanism

iv. Results

v. Human Practices

Introduction

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*10¹³ human cells but also 3.8*10¹³ bacteria. That means the mass of bacteria reaches 0.2 kg. In other words, humans are not solely composed of human cells. However, 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 vitro conditions. Therefore, we decided to establish co-culture system between human cells and bacteria.

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. If you can co-exist with photosynthetic bacteria or nitrogen fixing bacteria, you can photosynthesize or produce protein from air. If you could co-exist with bacteria, you could be a super human. We named this new type of human 'Coli Sapiens.'

Goal and Approach

Our original goals are as follows:

Mechanism

We established the following two systems.

Signal transduction system from bacteria to humans

~ Integration of systems derived from bacteria and humans ~

In this signal transduction system, the transcription level is controlled by integrating quorum sensing (bacterial cell-to-cell communication) and NF-kB, transcription factor in mammalian cell. We used this system the signal transduction from bacteria to human cells.

▼ Click here for more detail

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. 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 Trans Activation Domain (TAD) activates CMV minimal promoter and the expression of the downstream gene is induced. As a result, iP is produced and diffuses out of the cell.

Signal transduction system from human cells to bacteria

~ Integration of systems derived from bacteria and plants ~

In this signal transduction system, the transcription level is controlled by integrating signal transduction systems derived from bacteria and plants. We used this system the signal transduction from human cells to bacteria.

▼ Click here for more detail

Step1: AHK4 binding to iP performs an autophosphorylation reaction, transferring a phosphoryl group from ATP to a histidine residue of Histidine kinase (HK) domain. AHK4 transfers the phosphoryl group to its own internal receiver domain.

Step2: The phosphoryl group of AHK4 is transferred the histidine-containing phosphotransmitter, RcsD.

Step3: The phosphoryl group of RcsD is transferred the RcsB and RcsB is activated. Activated RcsB and another RR, RcsA form a hetero dimer and bind to cps operon promoter (which controls the production of polysaccharides). This series of phosphoryl group transmission is called the His-to-Asp phosphorelay (Read AHK4 Assay page).

Step4: The transcription of the gene downstream cps operon promoter is activated. In our genetic circuits, the gene downstream cps operon promoter is mazF. mazF is the gene of a toxin-antitoxin system. Please read Modelling page about the details of toxin-antitoxin system and mazF.

Co-culture system

We conducted experiments to validate the two systems above. Based on the results, we virtually integrated the two systems and conducted population change simulations.

Go to Modelling Page

Results

TraI Improvement

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 3-fold and it has been possible to induce iP synthesis in human cells from an early stage of E. coli's growth.

Chimeric Transcription Factor

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.

The term “Cont” means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.

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.

Cells were grown at room temperature on LB agar plates with and without iP. β-galactosidase activity was monitored by X-gal. Photographs were taken after 25h incubation.

Simulation

We again simulated the whole co-culture system using the parameter from assay data. The simulation showed that human cells have potential to control the population of E. coli, and both population settle to an appropriate ratio. In Fig. 7, u means the number of human cells and f means the flow rate.

Human Practices

From our full year experience in iGEM, we realized the necessity of verifying from a different point of view. In other words, we realized that we researchers ourselves must also continuously reflect on the risks and costs & benefits of the science we discover. In the workshop that we attended as our initial activity in iGEM, we learned from social scientists, the danger of grounding on the deficit model, which fixes on the idea that the general public is ignorant, and the importance of the two-way dialogue between society and researchers.