The first aim of my final project is to test either:

a) The viability of a codon-optimised adhesion protein to essentially act as a glue between the heart sleeve and the patient’s heart. To do this I will culture engineered bacteria. Two slides will be prepared: the first will have the hydrogel with the E. coli which have been genetically engineered to express an adhesion protein, and the second will have the hydrogel with E. coli - this will function as a control.

b) The signal transduction and biosensing ability of E. coli in the lab using sfGFP in a genetic circuit - with fluorescence being the indicator of a positive result. To do this I will genetically engineer E. coli to produce sfGFP in response to distress. I will run a small current through the hydrogel using a battery to simulate the pulse of the heart. This is to test the likelihood of the biosynthetic heart sleeve’s ability to detect the heart’s natural rhythm which it would adjust to in real time.

In order to do this I will be using Benchling and Twist Biosciences to design and synthesise DNA which will be tested in person at LifeFabs Institute.

Protocols

a) Adhesion Proteins

Here is the link to my Benchling for the adhesion protein plasmid

b) Biosensor Genetic Circuit

Here is the link to my Benchling for the biosensor genetic circuit

Once I had conducted the necessary preliminary experiments that I outlined above I would redo them many times and optimise the materials. For instance, I could design and test many adhesion proteins and rank these according to their stickiness but also their potential therapeutic viability and cost etc. Afterwards, I would move on to designing my own viable biotransistor scaffold - that is the very Living Heart Sleeve. I would test this many times and with mammalian cardiomyocytes. Ideally the scaffold should be relatively thin to fit around the heart but also quite robust so as to withstand being inside a body, so finding the right thickness will be a challenge. The end goal of this would be to bioprint the sleeve and test it with an animal heart (sorry cow or pig ❤️), probably a pig’s as their hearts are remarkably similar to human ones. Pigs are one of my favourite animals 🐷. I am not sure if it would be inserted into cows/pigs with heart conditions or if it would be tested on the hearts of cows/pigs who have already died.

The long term vision is to bring about a paradigm shift in VADs and cardiac therapeutics broadly. As I explained above the Living Heart Sleeve has the potential to reduce the risks of blood clotting and tissue rejection, pump and adjust to the patient’s natural heart rhythm, eliminate or at least lessen the need for medication, and also bring about the regeneration of a patient’s heart cells.

I have touched upon this above, but I must reiterate, that nothing like this exists as of yet. This technology would bypass the need for mechanical rotors made of titanium as in the case of traditional VADs and would be safer in the body.

I hope for the the Living Heart Sleeve to be used by cardiac patients with cardiovascular disease and arrythmia, for instance. I would hope that it would become viable for prophylaxic purposes in both regular, gerriactric, and pediatric contexts, thereby ultimately helping to prevent the incidence of heart attacks in vulnerable people. This hydrogel biotransistor technology could also have wide ranging implications for other fields.

Interpenetrated hydrogel transistors, showing their exclusive ability to resemble the spatial 3D structure of neural circuits in the brain, subjected to strain values of 0% and 30%. Video courtesy of HKU

I then read the paper published in Science: Increasing the dimensionality of transistors with hydrogels (Liu et al. 2025) by researchers at Hong Kong university (HKU), demonstrating the fesability of creating millimetre thick semi-conducting layers using hydrogels which are biocompatible, and became very interested in developing a Ventricular Assistive Device (VAD). The paper is unfortunately behind a paywall and is not even accessible through UCL alumni institutional login 💔, or via SciHub due to its being published after 2021. In fact, it was published on the 20th of November 2025, so not even 6 months ago from now. I had to get my brother at MIT to send me the PDF 🎻.

This paper details a breakthrough in material science as scientists at HKU document their ability to construct a 3D hydrogel which can achieve millimetre-scale 3D modulation. Traditional transistors which form the basis for modern electronics are 2D and hard, which severely limit their integration into biological systems. Whereas hydrogels are soft and biocompatible.

Scalable produced 3D hydrogel semiconductor fibers

Intrinsic stretchability of the hydrogel transistors

3D hydrogel transistors function similarly to organic electrochemical transistors (OETCs), however the OETCs lose functionality as their thickness increases. “In contrast, the linear thickness-capacitance dependency of hydrogel semiconductor remains consistent regardless of thickness (up to millimeters), indicating that complete 3D modulation is achieved in the hydrogel semiconductor” (Liu et al 2025:826). Here at HKU, the scientists were able to achieve this incredible modulation, balancing electron conductivity and ion activity whilst mimicking real neuronal connections through the pioneering of a double-network hydrogel system - something which creates structual stability.

Something which I found particiularly interesting was that the researchers used the reservoir computing (RC) framework (a machine learning algorithm which requires only minimal computational requirements and can operate with small training datasets) to demonstrate the potential for their 3D hydrogel transistor in the construction of neuromorphic circuits. The system they created achieved a prediction accuracy of up to 91.93%, which is comparable to conventional artificial neural networks! Moreover, this prediction accuracy can be maintained under up to 30% strain applied in any direction (see video above).

The authors also deal with the challenges of scaling up this new technology, stating that they believe the potential of 3d hydrogel semiconductors can only be fully leveraged through enabling efficient production using accessible and low-cost methods such as printing and textile manufacturing methods. They were able to develop a one-step water-processable fabrication protocol wherein the required materials for assembling the composite hydrogel semiconductor are premixed in a single step. The 3D hydrogel semiconductor can then be formed through a simple cross-linking process. Thankfully the necessary materials (with their sources) and methodology are detailed in a brilliant supplementary document.

Supplementary paper

Modulation Strategy

Transistor up close

Circuit

Intrinsic stretchability of the hydrogel transistors

The second article I read, which incidentally was cited in the first, is another published in Science: Transistor in a tube: A route to three-dimensional bioelectronics (Pitsalidis et al. 2018). Thankfully I was able to access this paper via SciHub which you can access by clicking the previous link. This paper basically lays down the foundations for flexible hydrogel transistors.

DNA Quality Control: Prior to synthesis with Twist Biosciences, verify with Juan Diego the complete 903 bp genetic circuit on Benchling to ensure the inclusion of the intact precA promoter, ribosome binding site (RBS), sfGFP reporter gene, and Mam7 adhesion sequence.

Quality Control Guardrail: Confirm that the sequence does not contain isolated restriction fragments like EcoRI (GAATTC) or HindIII (AAGCTT) sent during design iterations, ensuring the full “light bulb” coding region is present.

Primary Transformation: Thaw chemically competent E. coli DH5-alpha cells on ice, introduce 2 $\mu$L of the pTwist Chlor High Copy plasmid, and perform heat shock in a water bath at 42°C for exactly 45 seconds.

Outgrowth Phase: Incubate the shocked cells in SOC media at 37°C for 1 hour in a shaking incubator to allow expression of the chloramphenicol resistance marker.

Selective Plating: Spread 100 $\mu$L of the culture onto LB-Agar plates supplemented with Chloramphenicol and incubate overnight at 37°C.

Secondary Expression Strain Prep: Isolate successful plasmids via miniprep and repeat the transformation protocol into E. coli BL21(DE3) to maximize downstream sfGFP protein expression during the simulation.

Expected Results: Growth of distinct, well-isolated chloramphenicol-resistant colonies on the selective plates, verifying successful uptake and maintenance of the complete 903 bp biosensing plasmid backbone.

Bacterial Harvesting: Inoculate a 5 mL liquid LB-Chloramphenicol starter culture with a single colony of transformed BL21 cells and shake overnight at 37°C.

Cell Paste Concentration: Pellet the overnight culture using a laboratory microcentrifuge, decant the supernatant fluid completely, and resuspend the cells in a minimal volume of M9 media to form a highly concentrated, dense bacterial paste.

Hydrogel Preparation: Liquidize a biocompatible hydrogel matrix (such as Alginate or GelMA) using a hot plate, then allow it to cool down to an incubator temperature of 37°C to 40°C to guarantee bacterial cell viability during mixing.

Homogeneous Mixing: Pipette the dense cellular paste directly into the warm liquid hydrogel and stir gently with a sterile pipette tip to distribute the biosensors evenly without creating air bubbles.

Sleeve Molding: Cast the bacteria-loaded hydrogel mixture into a custom rectangular silicone mold containing pre-positioned, inert platinum wire electrodes at opposite ends.

Matrix Cross-linking: Complete the gelation process (via chemical cross-linking with Calcium Chloride for alginate, or light-directed cross-linking for GelMA) to anchor the bacteria within the structural web of the synthetic sleeve via expressed Mam7 outer-membrane proteins.

Expected Results: A structurally sound, solid-state hydrogel sleeve with an even distribution of living bacterial sensors structurally anchored to the polymer matrix, showing no signs of premature cell death or material deformation.

Chamber Setup: Secure the completed biosynthetic sleeve inside a sterile testing container and submerge the matrix slightly in ionic M9 minimal media to facilitate electrical conductivity while introducing mild nutrient stress.

Hardware Integration: Connect the embedded platinum electrodes to an external DC power supply regulated by an Arduino microcontroller programmed to deliver cyclic pulses.

Physiological Simulation: Initiate an electrical pacing routine delivering 1.5V to 2V pulses at a physiological human heart rate of 60 to 100 BPM (equivalent to a frequency of 1 Hz to 1.6 Hz) continuously for 60 minutes.

Baseline Measurement: Document the initial state of the sleeve using a digital camera and a 470 nm blue-light transilluminator equipped with an orange emission filter to confirm the absence of pre-stimulation fluorescence.

Kinetic Data Capture: Capture fluorescence images every 10 minutes during the heartbeat simulation run to monitor the progression of transcriptional activation.

Quantification: Read final endpoints on a fluorescence plate reader to calculate the overall signal transduction efficiency and dynamic range of the living wire.

Expected Results: Localized electrical current from the simulated heartbeat will induce cellular stress pathways within the matrix, prompting the precA promoter to drive robust transcription of sfGFP. This will manifest as a visible, quantifiable green fluorescence emission developing within 30 to 60 minutes near the electrode interfaces, confirming successful signal transduction!

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