What we expect from Experiments and Modeling:

For a proof of concept, we had to know some details about MagicBlock.

Velidation of Quorum Sensing Receivers

Used as test standards for other parts, The Fluorenscent response of QS receivers had to be examined first.

Choose Quorum Sensing systems with high orthogonality

Quorum Sensing systems' crosstalk problem in working conditions may led to functional failure for Bioblocks with greater than one QS systems.

Inter-Bioblock communication

Could bioblocks successfully communicate with each other?

Working parameters and kinetic model for MagicBlocks

How much time is needed for a bioblock to finish its task and when should the system proceed to the next layer? How much supernatant should be transfer to the next MagicBlock? To answer these questions, we had to build up a model and determine parameters of the model from experiment data.

Velidating Fluorescent QS receptors

We tested Fluorescent response for LasR-GFP, RhlR-GFP and RpaR-GFP BioBlocks using their cognate AHLs.

Fig. 1 Fluorescent response of LasR-GFP to 3OC12-HSL

Fig. 2 Fluorescent response of RhlR-GFP to 3OC6-HSL

Fig. 3 Fluorescent response of RpaR-GFP to Coumaroyl-HSL

Orthogonality prediction and test

Alone with literature search, We've used Molecular Docking to predict HSLs' affinity to different QS receiver proteins. We've choosed Las system from P. aeruginosa and RpaR system from R. palustris —of which predicted to be relatively orthogonal, to construct Advanced Bioblocks in our system.

Fig. 4 Structure of LasR and RpaR

We've also tested this prediction after plasmid construction:

Fig. 5 Orthogonality test of LasR-pLas-GFP (i):Fluorescent response to cognate and non-cognate AHLs (ii)Dose-Response curves for cognate and non-cognate AHLs (iii-vi)Fluorescent response to non-cognate AHLs in compared with 3OC12-HSL

Fig. 6 Orthogonality test of RpaR-pRpa-GFP (i):Fluorescent response to cognate and non-cognate AHLs (ii)Dose-Response curves for cognate and non-cognate AHLs (iii-vi)Fluorescent response to non-cognate AHLs in compared with Coumaroyl-HSL

Demostration of Inter-Bioblock communication.

To answer this question, we measured AHL production of Generator bioblocks using LC-MS and Fluorescent QS receptor array. Both method confirmed the generation of AHLs, and the quantitive result gives a conclusion that AHL production from a MagicBlock is enough to stimulate another block and actives it's gene expression under our system's working condition.

Because LC-MS can only indicate the production of our signal converter approxmately, but this data is too rough to instruct our following work. We get the quantitive relation between input signal concentration and output signal concentration by following experiments and deduction from model which can also indicate an unknown output signal concentration.

Kinetic model of MagicBlock

After expression experiments been conducted, our data is enough to describe the kinetics properties of a bioblock. We've modeled the response and re-generation of AHLs in typical intermediate bioblocks and determinated parameters for liquid handling robot we used for MagicBlock assembly.