Week 1 HW: Final Project Ideas

Biomanufacturing the “Universal Bio-Ink” for Autonomous Lab-on-a-Chip Diagnostic Sentinels (1) What you would like to biomanufacturer and why?

I would love to biomanufacturer “SynBio-Ink,” a programmable, cell-free diagnostic matrix containing lyophilized LAMP (Loop-mediated isothermal amplification) reagents, CRISPR-Cas effectors, and biosensor reporters.

My motivation is to be the part in solving global health and pandemic preparedness over the next 10 years. It will be based on decentralized and autonomous bio-surveillance. I dream a future where robotic “sentinel” devices homes use a near to universal cheap microfluidic Lab-on-a-Chip (LoC). For achieving that we need to bio-manufacture a bio-ink that can be printed directly onto these chips. We can create a universally deployable diagnostic fabric. This ink would allow a single chip to perform multiplexed LAMP amplification and CRISPR-based nucleic acid detection for hundreds of pathogens simultaneously.

(2) What living system would you use?

Rather than a traditional whole-cell factory, I would use a Cell-Free Protein Synthesis (CFPS) system derived from engineered Escherichia coli extract.

Inputs:

For establishing this kind of system we would require optimized E. coli lysate (containing ribosomes and transcriptional/translational machinery), energy buffers, amino acids, and synthetic DNA cassettes encoding customized CRISPR-Cas12/13 enzymes, Bst polymerase (for LAMP), and electrochemical reporter proteins.

Biological Process:

The Cell Free Systems would expresse the diagnostic proteins in vitro. This would allow us to bypass the cell membrane barrier and toxicity issues. The resulting soup would be combined with specific LAMP primers and guide RNAs.

Outputs:

We expect the output to be a fully functional “SynBio-Ink.” This cell-free matrix is then lyophilized (freeze-dried) directly onto the microfluidic channels of the diagnostic chip. When a liquid patient sample (e.g., saliva) is injected into the chip. The LAMP enzymes amplify the target viral/bacterial DNA, the CRISPR-Cas system detects the amplicons, and the collateral cleavage of reporter molecules triggering the biosensor.

(3) How would you use automation?

I would use an acoustic liquid-handling robot Echo 525 to automate the assembly and printing of the bio-chips. I would automate the acoustics dispensing of the Syn-Bioink into arrays on the substrate. This step is necessary because printing a multiplexed chip requires precision and micro-volumes that are impossible to achieve via manual pipetting.

(4) How much would you need to make, and how long would it take? (a) How much? Scale: Manufacturing SynBio-Ink to supply 1 pilot project for a global bio-surveillance network.

• A multiplexed microfluidic chip requires roughly 10 microliters (10 µL) of the cell-free diagnostic ink to populate all its reaction chambers.

• 1,000chips × 10 µL/chip = 10,000µL = 0.01 Liters of optimized SynBio-Ink.

(b) How long?

Producing 0.01 Liters of cell-free extract and printing 1000 chips could be achieved in 1 Day.

Culturing the source E. coli, lysing the cells, and preparing 0.01 Liters of continuous-flow CFPS extract can be completed in standard industrial bioreactors in 1-2 Days.

Using automated robotic factory floor with 10 parallel acoustic dispensers printing 1 chip per second each, printing 1,000 chips would take approximately 1-2 hours of continuous robotic operation.

Less than a week to run a pilot project with 1000 autonomous diagnostic nodes.

(5) What don’t you know yet?

The 3 biggest technical unknowns in this 10-year vision are:

  1. Can a complex matrix of cell-free lysate, Bst polymerase, and CRISPR-Cas enzymes be lyophilized onto a microfluidic chip and remain viable at room temperature for months?
  2. Will raw, unpurified patient samples (blood, saliva, or environmental wastewater) introduce inhibitors that stall the cell-free reaction, or will the chip require a complex, automated sample-prep module upstream of the sensors?
  3. I do not yet know if standard electrochemical biosensors are sensitive enough to detect this reduced signal without resulting in false negatives.