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.

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.