Introduction

During brainstorming, we brought up a lot of creative solutions, which the world should have but unafortunetely is stll not ready for them. Hence we devoted our attention to a more present concern: tissue-engineering. In this area of expertise, a three dimensional scaffold for the cells to attach is, amongst other things, essential. Since the origin and construction of these matrices is still a problem, new solutions are required. Currently, most of the preparations work in layers (for bone and cartilage material) or use nano fibers and textile technologies to generate a scaffold [1].

When thinking more about this problem we thought of a quite new approach on building three dimensional structures. What if we could just print tissue, bones or other stuff? How could we do that?

One of us had the idea to try something like the subsurface engraving of glassblocks, only instead of breaking small glass strucutres we wanted the bacteria to produce certain products like color [2]. So how can we activate our bacteria with laser, that they express a protein but would not burst into thousand tiny pieces?

Because of the seemingly limitless opportunities in Optogenetics, it was chosen as Method of the Year 2010 by Nature Methods and is titled as one of the breakthroughs of the decade [3]. The hallmark of optogenetics is introduction of fast light-activated channels and enzymes that allow temporally precise manipulation of electrical and biochemical events in bacteria. One can use channels derived from bacteriorhodopsin, photosynthesis associated complexes, like phycobilines in algae (for our red light sensor) or use existing light sensory domains already in e.coli (like our blue light promoter). Thus optogenetics provides for us an elegant way of converting a light signal in protein expression.

When thinking more carefully about the idea, we knew that we would need to immobilize the bacteria somehow to make sure that we can target certain spots. For this reason we have chosen a matrix named gelrite which can be passed through light with only little refraction. In addiotion to this fact it only contains a minimum of nutrient to enable and regulate growth and thus protein synthesis.

To achieve bacterial constructed three dimensional structures by using light induced gene expression it was necessary to come up with a precise regulation mechanism. Our most convincing approach was to design a logical AND-gate which converts two inputs in one output. In our case, it has been suitable to use two different wavelengths as inputs to induce gene expression, the output. Only when a bacterium is illuminated by both wavelengths it expresses a protein, e.g. a coloured molecule, to generate a three dimensional picture inside the gel block. Our AND-Gate is build up on light sensor systems developed and optimized by Edinburgh's iGEM-Team from 2010 and on recent results of the Voigt lab at UCSF [4].

This logical gate developed at UCSF is based on an amber stop-codon suppression through the non-canonical tRNA supD. A light sensitive promoter induces the expression of mRNA with a stop-codon suppression coding for a T7-polymerase, which can only be translated by ribosomes if the correct amber tRNA is present. The tRNA is expressed by a second light-sensitive promoter susceptible to another wavelenght. Only if both signals are present, the expression of a protein under the control of a T7-promoter takes place. We chose blue and red light promoter because their wavelengths differ significant from each other. Because of this construction we should be able to induce expression at desired spots only.

Our vision beyond the proof of principle of this project could be to fixate bacteria inside the gelrite which could express collagen and hydroxyl apatite instead of a dye,. Following we can generate bone material directly in the block without any further scaffolds. After protein synthesis and secretion one could simply peel off the gel and get a bone imagined and “drawn” with the light.

Since easy artificial production of human tissue and bones is still a difficult task, we think this approach could be a way to overcome certain problems in the field of tissue engineering. Assumed that all bacteria left over have been removed or at least destroyed. We would expect less immunrejection because the material does not show any surface antigens as other mammalian material does. Subsequently, the bone could be coated with cells of the recipient and a complete bone structure could have been created.

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