Interdependence

The goal of the interdependence project is to explore the use of amino acid auxotrophy as the framework for a mutually beneficial relationship between two different organisms, namely engineering bacteria to export essential nutrients in a readily available form and in the proper quantity to be sufficient for the host. This will create a strong dependency in a host lacking the production facility of these nutrients. We will perform the experiments on free-living cells, and investigate if the exported amino acids would be sufficient to sustain a S. cerevisiae host.

As part of the interdependence project the following will be examined:

• the functional expression of amino acid exporters in E. coli;
• the level of amino acid produced and exported by the symbiont;
• the effect of nutrient export on E. coli cell viability and growth;
• Grow auxotrophic S. cerevisiae in co-culture with respective amino acid exporting E. coli.
• In silico modelling growth of S. cerevisiae under the levels of amino acids that can be exported by E. coli to investigate the minimum number of E. coli symbionts to sustain an auxotrophic yeast host cell.

Number control

The goal of the number control project is to lower or stop symbiont replication in case of a high symbiont abundance and/or a starvation status of the host cell, with proof of concept being performed in E. coli cultures. To intertwine the cell replication cycle and lower the stress created by the presence of a symbiont inside the cytoplasm of the host, we aim to put the number control system under control with three signals: the symbiont abundance (a quorum sensing circuit), the host cell starvation status, and the host cell replication. QS and cell starvation should lower symbiont replication, while host replication should increase it. We aim to establish a replication control and intertwinement using a modular system based on CRISPR/Cas9 technology. A catalytically-dead Cas9 (dCas9) lacking endonuclease activity and a small guide RNA will be guided via RNA-DNA interaction to the origin of replication on the bacterial chromosome, to efficiently and transiently inhibit the chromosome replication (Wiktor J. et al, 2016).

The dCas9 system will be put under control of the quorum sensing pathway with the lux promoter, which will be active during high symbiont density. Additionally, we aim to put the number control system under control of cell starvation status and host cell replication. Moreover, to overcome an unrestrained cell growth, we aim to inhibit the membrane production silencing the expression of a key enzyme for lipid biosynthesis, i.e. Enoyl Acyl Reductase, commonly target of bacteriostatic drugs.

As part of the Number Control project the following will be examined:

• The effect of dCas9 expression on the cell cycle of E. coli
• The effect of sgRNA binding to various site of the chromosomal origin of replication on the cell cycle of E. coli
• The effectiveness of the two parts, i.e. dCas9 and sgRNA, to block DNA replication and cell growth
• QS efficiency in controlling dCas9 expression
• Inhibition of Enoyl Acyl Reductase expression via dCas9 in combination with a sgRNA targeting the enzyme promoter.

The integration of the host signals, i.e. the host starvation status and the cell cycle phase, to control the dCas9 expression will be tested using an in-silico model.

Protein import

The goal of the protein import project is to import fluorescent proteins into a bacteria by covalently connecting the protein to a cell-penetrating peptide (CPP).

CPPs are small peptides (down to 8 amino acids), generally rich in arginine molecules, capable of initiating cellular uptake of a large variety of molecules and proteins by inducing endocytosis. In both plant and mammalian cells CPPs have been shown to mediate protein uptake by both covalent and non-covalent association, with increased specificity during covalent association. However, in bacteria only non-covalent uptake of proteins have so far been demonstrated (Chang et al. 2014). We will explore the utilization of the synthetic CPP nona-arginine (R9) as a vector for facilitation of targeted protein import in bacteria.

As part of the Endocytosis project the following will be examined:

• An initial screening process aimed at evaluating the ability of a variety of bacterial species to take up fluorescent proteins through covalent and non-covalent CPP association
• Investigation of the cellular localization of proteins taken up in a CPP-mediated fashion.
• If proteins taken up are localized in membrane derived vesicles we will further investigate the potential ability of last 20 amino acids of influenza virus hemagglutinin (H2) to facilitate vesicular escape.

Proteins needed for this sub-project will be expressed with His-tags in E. coli and purified using affinity chromatography. Both flow cytometry and confocal microscopy will be used to evaluate bacterial protein uptake through use of the fluorescent proteins YFP and BFP as previously described (Chang et al. 2014). A lipophillic dye capable of staining membrane-derived vesicles (e.g. FM4-64) will be used to investigate cellular localization. To facilitate cleavage of R9 following import, either an endogenous or heterologously expressed peptidase will be used. Successful cleavage will be assessed through use of proteomics (e.g. targeted proteomics). The exact details for evaluation of enzymatic ability following protein import have still not been elucidated, we are however currently contemplating using an enzyme whose activity can be monitored through a colorimetric assay (e.g. 𝜷-lactamase).