1. Transcriptional light switch for cell adhesion

Using the blue light inducible promoter PBlind , we aim to use blue light (6hr, 475nm) to induce the transcription of Intimin, a cell surface protein, fused to either SpyTag (13 amino acids) or SpyCatcher (138 amino acids, 15kDa). SpyTag and SpyCatcher are two binding partners that come from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. SpyTag contains a reactive aspartate that forms a strong isopeptide bond with a reactive lysine residue in SpyCatcher when in close proximity.

Once a mixture of cells are light induced to display SpyTag or SpyCatcher on their cell surface, they are expected to aggregate. Similar to lithography, structures would be generated layer by layer from a flat surface by exposing a particular radius with blue light.

Designers note: after analysing multiple cell surface display proteins, we opted for Intimin (EaeA intimin) as it has shown to display passenger proteins of similar size to SpyCatcher and it had not been characterised by iGEM teams before. Other cell candidates considered include: ice nucleation protein, PgsA and OmpC.

Designed Experiments

A) Represents two cell lines expressing either intimin’-SpyTag or intimin’-SpyCatcher fusion proteins. Cells harbouring either construct under the control of a constitutive promoter would have either been mixed together or mixed with WT cells (control) (15min) or not mixed and passed through a particle sizer with a range of aperture sizes (0.4µm - 40µm) to compare aggregate sizes. As secondary experiments and for visual purposes we would have also incubated cells expressing Intimin-SpyTag with purified GFP-SpyCatcher, Intimin’-SpyCatcher with purified GFP-SpyTag and Intimin-GFP-SpyTag with Intimin-SpyCatcher cell lines for 15min and visualize cell-protein binding and cell-cell interaction via fluorescence microscopy. We also included a His-Tag within all SpyCatcher construct variants to enable in-vitro analysis of complexes formed with SpyTag variants.

B) Represents two cell lines expressing either intimin’-SpyTag or intimin’-SpyCatcher fusion proteins pre and post light induction. Aggregation experiments described in A) would have been repeated after replacing the constitutive promoter with PBlind , blue-light inducible promoter and transforming cells with EL222 (blue light inducer) under a constitutive promoter (C). Cell aggregation and fluorescence experiments would have been carried out with cells grown overnight under darkness or under blue-light and in the presence or absence of the EL222 construct.

2. Post-translational light switch for cell adhesion

As a more immediate induction of cell aggregation, we aimed to develop a post-translational light switch in which cells are constitutively expressing either Intimin’ fusion proteins with SpyTag or an inactive version of SpyCatcher. Only upon light exposure, SpyCatcher is able to bind SpyTag resulting in a much faster response than transcriptional induction. This is achieved by incorporating a photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine, in place of the reactive lysine in SpyCatcher required for the covalent bond formation with the SpyTag aspartate residue. Upon exposure to UV light (20min, 365nm), the “cage” group in the unnatural photocaged amino acid is cleaved off revealing the native amino acid and a biologically active protein. This approach also adds a layer of bio-containment as cells will only function when externally supplied with UAA.

To achieve this, we introduced an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher. Amberless E. coli cells also have to express pyrrolysyl tRNA (pyIT/tRNAPylCUA) from M. mazei and pyrrolysyl-tRNA synthetase from M. barkeri and the UAA must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. The orthogonality of this system has shown to work in both E. coli and mammalian cells.

Figure 2. Post-translational light induction of cell aggregation. A) Represents two cell lines expressing constitutively either intimin’-SpyTag or the photocaged version of intimin’-SpyCatcher fusion proteins. Cells harbouring either construct would have either been mixed together or mixed with WT cells (control) or not mixed and exposed to UV light (365nm) or not exposed for 25min-1hr and then passed through a particle sizer with a range of aperture sizes (0.4µm - 40µm) to compare aggregate sizes. As secondary experiments and for visual purposes we would have also incubated cells expressing Intimin’-SpyTag with purified photocaged GFP-SpyCatcher, photocaged Intimin’-SpyCatcher with purified GFP-SpyTag and Intimin-GFP-SpyTag with photocaged Intimin-SpyCatcher cell lines pre and post photo-lysis (365nm) for 25min-1hr to visualize cell-protein binding and cell-cell interaction via fluorescence microscopy. We also included a His-Tag within all photocaged SpyCatcher construct variants to enable in-vitro analysis of complexes formed with SpyTag variants.

3. Polymer production/binding

Bacteria has been widely used for the production of biomaterials to generate sustainable and eco friendly bricks, bioplastic products, items of clothing as well as “living materials” by incorporating nanoparticles into biofilms. With Barchitecture we can light-induce cellular 3D structural arrangements while producing, binding or degrading biomaterials such as PHA or silicates.

Proposed design for the production, secretion and binding of PHA granules:

Through a similar mechanism, bacteria, could be light-guided to form precise and intricate structures that can then simultaneously produce and bind biosilica. This approach will enable “growing” electronics just with the guidance of a particular wavelength of light.

Our engineered cells would form the desired 3D structure using blue light. These cells would also express photocaged (inactive) recombinant silicatein on their surface. Another wavelength of light would activate silicatein to begin the production of biosilica from water-soluble biosilica precursors added to the media. Our cells could also present ligands on their surface to attach the produced polymer to their surface.

Our wet lab data

We tested whether the promoter has any significant leakage. Also, we wanted to show that GFP cannot be expressed in the absence of EL222. This is of particular interest as the aim of LIT is to demonstrate the versatility and high precision of light control. As shown in Figure1, only J23151-GFP (positive control) had a significant difference in fluorescence compared to R0040-GFP (negative control) WT cells and the Luria Broth (LB) in both dark and Blue-light conditions. Pblind-GFP had no significantly different fluorescence level compared to the LB baseline, negative control or WT cells in either condition. This is expected, as the EL222 protein is required for blue-light inducible transcriptional activation.

Figure 1. Blue light inducible promoter (Pblind) characterisation. J23151 is a constitutive promoter, R0040 is a TetR repressible promoter (repression inhibited only by the addition of tetracycline), Pblind promoter is a fusion of EL222 (photosensitive transcription factor) binding region and the luxI promoter, where EL222 is only able to dimerize and bind the Pblind promoter upon blue light exposure, where it can then recruit RNAP and drive the transcription of genes downstream. Wild type (WT) 10beta cells were transformed with J23151-GFP (positive control), R0040-GFP (negative control) or Pblind-GFP. All cells were grown overnight at 37°C in darkness or exposed to blue light (465nm) and diluted to OD600=0.6 to record GFP fluorescence. The data represents the mean of 3 biological replicates and 4 technical replicates for each condition. Luria broth (LB) was included as a baseline for the fluorescence. Error bars represent the SD and statistical significance of **** P < 0.0001 was calculated using the Tukey's multiple comparisons test.