Demonstrate

Introduction

The aim of our project is having human cells control the number of bacteria for a long period; in other words, we aim to create a sustainable co-culture system of human and bacterial cells. In our co-culture system, human cells repress bacterial growth moderately so that too many bacterial cells do not display negative influence to human cells. However, given that our co-culture system is supposed to be applied to human therapies, every effort must be made for bacteria not to show unexpected toxicity and survive in external environments. To this end, we selected and engineered two BioBrick parts which encode kill switches for biosafety of future applications.

Signaling molecules selection

The most studied signaling molecules that are used for intercellular communication in nature are AHLs (acyl-homoserine lactone). AHLs are produced by some kinds of gram negative bacteria and are used for regulating gene expression depending on population density; this mechanism is called “quorum sensing”. Therefore, we decided to use AHLs for human and bacterial cell communication, but toxic substances cannot be used as signaling molecules in our project.

It has been reported that AHLs stimulate human immune system [1], but toxic effects of AHLs have not been reported elsewhere. In order to gain insights on the toxicity, 3OC8HSL (hereafter C8), which is a kind of AHLs and is used as a signaling molecule in our project, was added into the culture medium of human cells in various concentrations. The result of C8 toxicity test suggested that C8 was nontoxic to human cells. The details are shown below.

C8 toxicity test

We evaluated the toxicity of C8 against untransformed EA.hy926, a human cell line used in our project, by conducting MTT assay following the protocol (at the end of this page). MTT assay is a colorimetric assay for assessing cell metabolic activity. It can be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. As shown in Fig. 1 and 2, it is confirmed that over 80 microM of C8 can be toxic for the cells. We concluded that the amount of C8 produced by E. coli will not be toxic for human cells and therefore will not disturb the communication between E. coli and human cells.

Fig. 1 Time course observation of the cells in MTT Assay

Fig. 2 Cell viability 24 hours after C8 treatment in EA.hy 926

Parts selection

Harmful parts and substances are not allowed in our project. Through enormous investigation of literatures, we assumed that chimeric transcription factor (RelA/TraR) and iP (isopentenyl adenine) can be synthesized by human cells without harming human cells [2]. TraR is a receptor protein of C8 as well as a transcription activating protein depending on C8 in Agrobacterium tumefaciens. RelA is a kind of NF-kB and is a human transcriptional factor that regulates a wide range of genes.

It has been reported that when TraR is fused with the transcription activating domain of RelA and the nuclear localization signal sequence, the chimeric protein RelA/TraR acts as a transcription factor on the tra box DNA sequence (TraR-binding sequence) in human cells. Importantly, this transcription has shown to occur depending on C8. iP is a kind of cytokinin. Cytokinins are the signaling molecules (Phytohormones) that plants produce and play important roles in plant cell growth and differentiation.

When the genes of RelA/TraR and iP synthesis enzymes (Arabidopsis thaliana AtIPT4 and LOG1) were introduced to human cells, no harmful effects like cell growth inhibition were observed (Read Chimeric transcription factor page).

Fig. 3 Human cells introduced the genes of RelA/TraR and iP synthesis

Fig. 4 Negative control (Not gene transuded)

Double kill switches

To stop bacterial excessive growth, we introduced two kill switches, AHK4 and MazF, into mammalian cells. AHK4 is a receptor of iP derived from Arabidopsis thaliana and MazF is toxic component of an Escherichia coli toxin-antitoxin (TA) module. As described in the Project page and the AHK4 Assay page, AHK4 is originally employed here to achieve intercellular signaling from mammalian cells to bacterial cells. However, during our assay, AHK4 was found to be toxic for E. coli and inhibited cell growth (Read AHK4 Assay). Therefore, we came up with an idea where AHK4 is exploited as a kill switch for E. coli after receiving iP and completing signal transduction. Since the promoter for expressing AHK4 is the arabinose-inducible type (BBa_K2505001) throughout our project, adding arabinose becomes a trigger for kill switch in E. coli. Moreover, we characterized that the downstream gene of cps promoter can be expressed by arabinose independent of AHK4 response(Read AHK4 Assay). Thus, by employing mazF as the downstream gene, both AHK4 and MazF can be easily induced by arabinose. These double kill switches are expected to work to repress harmful bacterial excessive growth.

Appendix: MTT Assay Protocol

Read MTT Assay Protocol page

Reference

[1]Inter-kingdom signalling: communication between bacteria and their hosts (2008) David T. Hughes & Vanessa Sperandio

[2]Foundational Platform for Mammalian Synthetic Biology (2012) Noah Davidsohn

Team:TokyoTech/Demonstrate