IGEM:Imperial/2010/Rapid Response Sensor System

Rapid Response system relying on pre-produced pigments or fluorescent proteins.

RAPID RESPONSE MODULE DAY 5 (9.07.10)
IMPERIAL COLLEGE 2010


 * IGEM:Imperial/2010/TCS and effector ideas

Emphasis on the foundational importance of this idea. Our rapid response module can be manipulated to have different outputs. We are aiming for a fast response but additionally a response in which we can have dynamic time control over the output. Use our module as a framework for other sorts of applications/ with different inputs and outputs.

Using linked 2 component systems we will create a logic gate type system with AND, OR functions. The effector protein in the response relay chain will activate expression of a protease which will induce sequestered pigment dispersal.

This system will have multiple colour outputs, at it simplest we can work with 3 coloured outputs responding to 2 different stimuli by using a combination of two pigments to make the 3rd colour.

The pre-made pigments or fluorescent proteins will be sequestered onto a scaffold protein via linking sequences. These will contain proteolytic cleavage sites unique to each coloured pigment. Proteases activated by a particular 2 component system will cleave off pigments of one colour. Therefore single or combinations of activated 2 component systems will produce a particular coloured pigment release.

The types of pigment that we've considered include bilins and phytochromes (or non fluorescent pigments that can be tagged), the family of fluorescent proteins and camgaroos.

An alternative to the scaffold sequestering idea is to create a poly-cistronic chain of linked pigments which can then be cleaved into single entities.

Use colourless to coloured response is the best approach. So two interacting proteins on the scaffold type molecule are colourless, upon cleavage the interaction is broken and colour is visualized.

Using inteins to activate an enzyme by-passing the transcription-translation step.

Fret based output system using quenchers as an alternative to bilins. Proof of principle techniques exist (caspase detection experiment)

Resources that may be helpful= BRENDA enzyme database.
 * Research into natural proteases that are activated by phosphorylation or an alternative which bypass the transcription step, and research into cleavage specificities and the enzyme activity ~(kinetics).
 * Research into different types of output e.g. BRET.

13.07.10

polyprotein (chain of pigments; bilins and phytochromes joined together by linking sequences containing protease sites)

vs scaffold protein idea, (refer to tom ellis lectures synbio 2010!) using SH2 SH3 PDZ binding domains within a central scaffold protein to bind SH2 SH3 PDZ domain linked pigments in a homologous way which sequesters the pigment in these large scaffold complexes. on the scaffold the pigments are either coloured or non-coloured and then upon release the pigment is dispersed and colour will be produced. The effector protein of the input to output 2 component system in this scenario could phosphorylate the binding domain of the SH2/SH3/PDZ to initiate binding of the pigments to the scaffold or vice versa (bind when tyr is phosphorylated, dissociate upon another stimulus- dephosphorylation.)

Use of inteins to activate the protease which may cleave the polyprotein, or pigments covalently attached to a scaffold. Small molecule activated inteins have been engineered. Artificial inteins contain a particular ligand receptor which renders the auto-catalytic intein inactive, upon a ligand binding its receptor it can induce the splicing event. this is the kind of switch event that would work in our system and in literature there are examples of very specific intein-small molecule systems which work but the ligand and receptor are very specific to whichever application the paper was working towards. like thyroid hormone or rapamycin. Papers suggest that using a particular intein (fast splicing variant of Mycobacterium tuberculosis RecA wildtype intein) can work when engineered into different proteins (i think?) The idea of making our own fusion protease plus intein engineered to respond to a ligand is too complex. In the papers the activating ligand is usually added. Bypass the two component system which would have activated an effector protein which induced protease-intein splicing and let the stimuli enter the bacteria and activate splicing directly.

intein paper mini review Use of a sigma 54 systems to ensure no basal transcription occurs- minimise noise to stop false positives.

HIV protease is 99 amino acids, a potential protease that wouldn't take too much time to transcribe translate if we cannot bypass that step.

really cool paper on molecular tools that we can perhaps use somehow