3-D Printing Bioreactors for Continuous Cell-Free Gene Expression

By Aubrie Hetherington

NSF REU at the University of Minnesota

Summer 2025

Cell-Free Gene Expression

• Cell-free transcription translation (TXTL) is an in vitro platform to synthesize RNA and proteins.
• Escherichia coli TXTL is the most common cell-free system, because of fast replication rates, affordability, and accessibility of engineering strains.

Gene Circuit Prototyping

Continuous Systems

• By feeding a TXTL reaction with energy molecules through a semi-permeable membrane it is possible to extend the reaction times from 10 hours to 2 days.
• The commercially available membrane systems are not ideal because:
• High cost (~$2000)
• Single-use
• Low throughput
• Incompatibility with standard fluorescence readers, which prevents temporal monitoring

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My Research Goal

• Create a continuous system plate that is:
• Cost-effective
• High-throughput
• Easily created
• Shareable
• Fluorescence reader compatible

Fused Deposition Modeling (FDM)

Bhattacharjee et al., 2016

Diffusion through the Membrane

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/separation-characteristics-dialysis-membranes.html

Diffusion through the Membrane

Constructing the Design

Semi-permeable membrane

Test Design

• We started simple with a 3-well design to perfect our lip feature
• This showed us:
• No glue on reaction site
• Tighter membrane reducing wrinkles
• Great alignment
• Still needing to use glue

Current Design

Alignment pins insure accurate alignment of both wells

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Lips tighten the membrane and prevent glue on the reaction site

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• 24-Wells allows for high throughput experiments
• Sized for compatibility with standard fluorescence readers

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Air funnels permit extra air to oxygenate the TXTL reaction.

Water Absorption

• FDM 3-D printing doesn’t produce watertight prints
• Prints layer by layer
• Infill is usually only 15%
• Could we lose our sample to absorption?
• Doesn’t seem so, but we plan to test again

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2 Layers

4 Layers

6 Layers

Glue Toxicity

• Through our design process, we have utilized two different glues.
• Loctite EA E-120HP was easier to apply and was stronger.
• JB Weld Marine Weld was tough to apply but also sealed well.
• We tested these glues with a TXTL reaction to determine if the glue was toxic to the reaction.
• Loctite (A & C) completely killed the reaction while JB Weld (B & D) seems to show no effect.

vs

https://www.jbweld.com/product/marineweld-twin-tube?srsltid=AfmBOooM8JC_6mpX803fAZXfCHo6Fp5m2V5waDBSN1wmqOU7-YvYU2qX

https://next.henkel-adhesives.com/us/en/products/industrial-adhesives/central-pdp.html/loctite-ea-e-120hp/BP00000164738.html

Kinetic Dye Test

• To confirm correct membrane usage, we measured the transport of different sized dyes across it over a 52-hour period.
• The membrane had a MWCO of 12-14kDa.

Aligning the Fluorescence Reader

• Standard fluorescence readers are manufactured for standard microplates
• Our design specifics and improvements required a custom alignment
• Using different concentrations of 40kDa Fluorescein, we centered the reader on each well

https://www.microplates.com/product/24-well-clear-bottom-ps-microplate-white-f-bottom-assay-plate/

My Research Completion

• Creates a continuous system plate that is:
• Cost-effective
• High-throughput
• Easily created
• Shareable
• Fluorescence reader compatible

Approximate Cost = $7

• Filament cost for one of our prints is $3
• Membrane costs $2 per design
• Glue costs $ 2 per design
• Printer is free to use
• If purchasing personal printer $1000

High-Throughput

• 24-well design with scalability allows for whatever throughput desired
• Simple to create and use multiple plates

Easily Created and Shareable

• Average time spent directly with design:
• <2 Hour
• Time to print:
• 14 Hours
• Glue Curing
• 15 Hours

• CAD file allows anyone with 3-D printing access to print it
• Simple process to glue
• Minimal time hands-on
• Lightweight for shipping to collaborators

Fluorescence Reader Compatible

• Sized for standard fluorescence reader
• Can be read from top of bottom

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Future Plans

• Progression to a TXTL reaction to confirm effectiveness
• Expanding to more wells to increase throughput
• 48-wells
• 96-wells
• Modifying membrane usage
• Reduce waste
• Reduce cost
• Eliminate risk of communication through membrane

Thank you!�

Acknowledgements:

Antoinette Barsoum

Kellen Grimes

Ava Scroggins

Aset Khakimzhan

Vincent Noireaux

Anderson Student Innovation Labs Staff

Bhattacharjee, N., Urrios, A., Kang, S., & Folch, A. (2016). The upcoming 3D-printing revolution in microfluidics. Lab on a Chip, 16(10), 1720–1742. https://doi.org/10.1039/C6LC00163G�

References