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Artificial Cells with Distributed Cores to Decipher Protein Function

Periodic Reporting for period 2 - ACDC (Artificial Cells with Distributed Cores to Decipher Protein Function)

Reporting period: 2020-02-01 to 2022-01-31

We envision a future where ‘chemical apps’ on mobile devices produce on demand valuable compounds for health and performance as well as apps for bioagent threat detection and disease. To take concrete and defined steps toward this future vision, we will exploit the miniaturization provided by lab on a chip technology and construct responsive architectures and metabolism based on living cells and tissues. We will build programmable and re-configurable, (bio)chemical processes, with precision, order, and as hierarchical cellular constructs, in the same way as living systems. We will enable microscale, liquid-based, chemical compartmentalisation (cores), and inter-compartmental (core-core) communication, just as one finds in organelles, cells and tissues. The ACDC project focusses on developing this next generation technology through a detailed workplan that heavily involves the nontrivial tasks of integrating diverse state of the art technologies including microfluidics, microwave resonators, DNA-based supramolecular assembly, in vitro gene expression and the integration of membrane channels into a functional platform.

The overall objective of the ACDC project is to manufacture compartmentalised, liquid-based chemistries, as discrete, yet interactive and multifunctional cores, within micro-scale containment capsules as communicating micro-laboratories. To achieve the ambition of the project, we have 8 overall objectives.

Objective 1. Produce multifunctional cores and containment capsules through use of 2D/3D printed, multiphase microfluidics.
Objective 2. Demonstrate core-core and core-environment communication together with energy harvesting.
Objective 3. Produce reconfigurable capsules based on DNA barcoding.
Objective 4. Build a compiler that translates formally described instances of chemical processes into a ACDC design.
Objective 5. Test our ACDC technology using small molecule library screening on targeted membrane proteins.
Objective 6. Evaluate ACDC emergent technologies and results for potential intellectual property for exploitation.
Objective 7. Execute targeted dissemination activities to public and stakeholders to enliven ACDC aims and Living Technologies in general
Objective 8. Generate a discussion about the societal and ethical impact of the technologies developed in ACDC and in Living Technologies in general.
Objective 1. Produce multifunctional cores and containment capsules through use of 2D/3D printed, multiphase microfluidics.
This continues to be a main research arm of the ACDC project. CU designed and manufactured 3D-printed microfluidic devices to produce ACDC capsules. These devices are being implemented with ELV pressure pump technology. Functionality continues to be developed with CU, UNITN and EXP. Core and capsule arrangement and interconnectivity continues to be developed in collaboration with ZHAW. CU also implemented control using microwave technology and droplet levitation. ELV is developing and beta-testing through the consortium a thermalization system composed of thermal reservoir holders and a chamber.
Objective 2. Demonstrate core-core and core-environment communication together with energy harvesting.
Demonstrations of thermal and ion triggerable ATPS platforms have been developed and switching is shown to be functional in ACDC cores using membrane proteins as transduction elements (CU) and microwave technology. Specific collaborative projects initiated during COVID lockdowns (Cholesterol and mini-modules focus groups) have enabled collaborative work with UNITN on perfringolysin-O (PFO) where techniques for assaying PFO cholesterol response are being developed. ACDC collaborative project with EXP has progressed and first measurements of an Explora-Cardiff jointly developed cargo protein are underway.
Objective 3. Produce reconfigurable capsules based on DNA barcoding.
UNITN executed the production of DNA-labelled capsules, with one step and two step labeling, exploiting 3D microfluidic chips printed in CU and UNITN, with ELV microfluidic flow controller and EXP DNA tags. UNITN is also developing the reconfiguration system based on in situ RNA synthesis. ZHAW designed an additional simulator that captures the dynamics of assembly.
Objective 4. Build a compiler that translates formally described instances of chemical processes into an ACDC design.
Considerable progress has been made in the simulation of multiple compartment systems. The challenge lies not only in the physical dynamics but also in the efficient treatment of several thousand particles. An important result is the definition of quantities which a) can be measured in an experiment, and b) are sufficient to characterize different types of vesicle/droplet assemblies.
Objective 5. Test our ACDC technology using small molecule library screening on targeted membrane proteins.
EXP designed multicistronic and single-gene plasmids, designed plasmids for the expression of proteins able to cross lipid membranes in collaboration with CU. CU determined appropriate core-core/capsule-capsule cargo delivery, reporting and sensing protein/peptide modules to be included in Explora plasmid construction and worked alongside Explora to determine potential and optimal fusion construct arrangements. UNITN completed preparation of the cell-free system, protein pores and generation of vesicles. ZHAW developed the simulator for the 3D structure of cores.
Objective 6. Evaluate ACDC emergent technologies and results for potential intellectual property for exploitation.
This is an ongoing effort in the consortium. See for example the market research in Task 1.6 and Innovation Radar documentation.
Objective 7. Execute targeted dissemination activities to public and stakeholders to enliven ACDC aims and Living Technologies in general
The outreach and training programme conducted by MUSE with ALL consortium members has been carried out. Most of the activities have been moved to an online or blended format, such has the Open Talks, Start-up School and Summer School.
Objective 8. Generate a discussion about the societal and ethical impact of the technologies developed in ACDC and in Living Technologies in general.
Much of this objective is scheduled to be activated in the second half of the project and is expected to be impacted by the state of the world due to pandemic reactive measures.
A report for this objective has already been submitted.
We have achieved progress beyond the state of the art with regard to several areas. 1. 3D printed fluidics, 2. reconfigurable systems, 3. chemical compiling. We are developing these new results which will eventually result in public access reports, publications, presentations, and public forum discussions. We expect scientific impact in the areas of soft-matter physics, complex systems, and custom small scale manufacturing. Given that this is only the first year of the project, there are many technical goals of the project yet to fulfill before socio-economic impacts can be realistically addressed. However given the progress so far along all research lines in the ACDC project, we fully expect to generate the technology and knowledge towards producing chemical apps for public use and distribution for user-defined on-demand needs.
ACDC core simulation
ACDC core assembly within capsules
ACDC capsule assembly