Introduction

During brainstorming, we discussed a lot of different problems. We came across tissue-engineering where you need, amongst other things, a three dimensional scaffold for the cells to attach. Since the origin and generation of these matrices is still a problem, new solutions are needed. Currently, most of the preparations work in layers (for bone and cartilage material) or use nano fibers and textile technologies to generate a scaffold.

When thinking more about this problem we thought of a different 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 engraving of glassblocks from the inside with bacteria. There you can print a picture inside glass with laser. How can we activate our bacteria with laser, so that they express a protein?

There we could use optogenetics which provides an elegant way of converting a light signal in proteinexpression. Optogenetics was chosen Method of the Year 2010 by Nature Methods and is titled as one of the breakthroughs of the decade. 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).

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 penetrated by light with only little refraction and which contains a minimum of nutrient to enable growth and protein synthesis.

To achieve 3 dimensional objects by using light induced gene expression it was necessary 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 hit by both wavelengths it should express a protein, e.g. a coloured molecule, to generate a three dimensional picture inside the agar 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.[1]

This logical gate developed at UCSF is based on amber stop-codon suppression via 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. Only if both signals are present, the expression of a protein under the control of a T7-promoter can start. We chose blue and red light promoter because their wavelengths lie wide apart. With this we should be able to target activation of expression in desired spots.

One step beyond the proof of principle could be to put bacteria inside the agar which could express collagen and hydroxyl apatite instead of a dye, so we can generate bone material inside the block. After protein synthesis and secretion one could simply peel off the agar and get a bone scaffold 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 washed away or at least have been destroyed, we would expect less immunrejection because the material does not show any surface antigens as other mammalian material do. Subsequently, the bone could be coated with cells of the recipient and a complete bone structure could have been created.

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