Input Modules

Inducible promoters

If the expression of GAL4 is powered directly by an inducible promoter, for example a phosphate-starvation inducible one, then activation of the processing module will be directly tied to this condition.

Other promoters induced by heat or salt stress, nitrate, sulphate or potassium starvation could all be found (See our promoter hunt page under the marchantia tab for how we looked for them) and turned into input modules.

Promoters and associated sensing systems in plants have naturally evolved to be hugely specific for plant-relevant challenges. Therefore most of the utility of harnessing endogenous promoters in our synthetic signalling cascade would crop up in plant-based applications such as monitoring soil conditions in agricultural earth or allotments.

Hammerhead ribozymes

A second type of input would be the hammerhead ribozyme aptamer system created by Christina Smolke's lab at Stanford. This consists of an RNA sequence made of two fused parts. One catalytically self-cleaving RNA molecule called a hammerhead ribozyme is fused with a ligand-binding RNA sequence called an RNA aptamer.

RNA aptamers specific for thousands of different molecules and ions have been generated, and a library of these can be browsed here http://aptamer.icmb.utexas.edu/)

By fusing an aptamer to the self-cleaving hammerhead ribozyme in an appropriate way, its self-cleavage activity can be modulated by the binding or absence of the ligand. In other words, when the ligand binds, it can promote or inhibit the cleavage activity by effecting a conformational change. In this way, the aptamer becomes an on or off switch for the ribozyme.

If the hammerhead-aptamer fusion is placed in between the GAL4 gene and a terminator, when switched off by the binding of its ligand of interest, it will stop cutting the poly-A tail off of the mRNA, resulting in increased gal4 expression. If the RNA fusion is configured differently, the binding of the ligand can alternatively become an on switch for the hammerhead ribozyme and therefore an off switch for GAL4.

Output Modules

These are how mösbi will let the user know that its input module has been activated. It can be anything that is detectable by human senses, be it a colour, a change in shape, a smell, or even a sound if an appropriate genetic system is found and a module designed.

We addressed the two easiest human senses to alert: Sight and smell. For visual alert, we engineered Marchantia polymorpha to express brightly coloured chromoproteins that are produced by corals and sea anemones and were brought to iGEM by Uppsala 2011.

We picked five chromoproteins:

AsPink
EforRed
TsPurple
AmilCP
AeBlue

then assembled two types of construct - one set with the N7 nuclear localisation tag (BBa_K1484104) attached, the others without.

The chromoproteins and chromoprotein fusions' expression was powered by the 35S promoter. To see how this went see our results page.

To create a module that uses smell as an output, we tried to make marchantia produce the smell of raspberries when the input module was activated. To achieve this we transformed with genes for the enzymes in this pathway:

To see how this went, also see our results page.