Phactory yields phages with toxicity levels that allow for oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice, while direct intravenous application requires additional purification steps. To overcome these hurdles, we prototyped two 3D-printed fluidic devices that each can be assembled for less than $5.

For oral application, we built the Phagecapsulator, a nozzle to encapsulate the phages in monodisperse calcium-alginate microspheres that protect them in the stomach. For intravenous administration, we can purify the bacteriophages from the remaining cell-extract via fractionation in a pressure-driven size-exclusion Phactory-Purificator filter system.

Additionally, we built microfluidic hardware for our human practice project OraColi .

Interactive Figure 1: Phage-Encapsulator
Interactive Figure 2: Phage-Purifier



As in many manufacturing processes, packaging of our produced phages is the final step of Phactory. Packaging provides long-term storability and ensures a clean and safe product for patients. Lacking access to commercial bioencapsulation devices such as the Inotech Encapsulator IER-50, available at $10,000, we engineered Phage-Encapsulator, an affordable device for encapsulating phages in monodisperse alginate spheres (1).

Requiring only standard parts, phage solution, alginate and compressed air, Phage-Encapsulator makes phage packaging accessible to non-specialized laboratories. The throughput is sufficient to produce encapsulated phages for oral application on-site in less than 2 hours.

Overall design

The core part of the Phage-Encapsulator consists of a dispensing tip that is continuously fed with phage containing alginate solution by a syringe pump. The dispensing tip is surrounded by a chamber connected to a compressed air supply to create a steady stream of air around the tip that shears off a jet of monodisperse droplets, smaller than the diameter of the tip. The alginate droplets are sprayed into a Calcium-Chloride bath where the rapid crosslinking reaction occurs.

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Explosion view of the Phage-Encapsulator.
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Slice view highlighting the nozzle design.
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Monodisperse Alginate Droplets at 150X magnification.

Individual Parts:

Alginate Solution

The negatively charged polysaccharide alginate is commonly used for cell encapsulation or as food additive. Alginate undergoes strong shrinking when the pH of the environment drops below its pKa (2). Due to its pKa of ~3.5 this is the case in gastric fluid with a pH of 1 – 2. These properties render alginate spheres an ideal delivery substrate, protecting the phages in the stomach and releasing them in the basic milieu of the intestinal tract. As a tradeoff between alginate viscosity and gel strength, we found that a mixture of 1.8% high viscosity alginate (Art.-No. 9180.1, ROTH) and 0.2 low viscosity alginate (A1112, Sigma Aldrich) represents a good compromise.


Our final design of the nozzle is shown in Interactive Figure 1. It consists of a chamber that can be connected to a pressure supply via a universial tube adapter. Consisting of two parts, the nozzle does not get in contact with the phage solution, so it can be reused. The dispensing tip can be easily inserted via a Luer-lock adapter and lines up precisely with the nozzle orifice. A centering aid is located behind the orifice to reduce variances in cannula alignment, while the air stream is not disturbed unequally by its symmetric shape. At a given air pressure, the orifice diameter determines the velocity of the air stream and consequently the droplet diameter.

Calcium bath

Rapid gelation of the phage-containing alginate droplets was obtained in a 10 mM MgSO4 solution with 1.8% CaCl2. Aggregation of alginate droplets was prevented by continuous stirring during droplet generation with a magnet stirrer.

Syringe pump

In our experiments we used a commercial syringe pump from TSE Systems (type 540060) to create a continuous alginate stream. We designed our nozzle to be compatible with other types of syringe pumps, including open-source solutions.

Pressure supply

As a pressure supply we used the house gas line, reduced to 1 bar. If not available, the universial tube adapter ensures compatibility to alternative pressure supplies, including portable, open-source solutions.

Part List

  • syringe (1ml Braun Omnifix)
  • dispensing Tip (ID 0.51mm, Vieweg)
  • syringe pump
  • pressure supply
  • pneumatic tubing
  • 4x M3x20 screws + nuts
  • 3D printed nozzle (PMMA)


Our results show that after 1 hour incubation in simulated gastric fluid, active phages are successfully released in simulated intestinal fluid. Within the limited time frame of the iGEM competition we were able to show storability of these alginate spheres with constant phage activity at 4°C for four weeks, while stability for several months was shown in the literature (3).

Droplets are Monodisperse

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Alginate composition of 1.8 % Alginate and 0.2 % low-viscosity Alginate delivers spherical Alginate microspheres.
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Alginate encapsulated phages, DNA stained with SYBR Gold.
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Monodisperse Alginate Droplets at 150X magnification.

In order to achieve defined phage concentrations and therefore defined doses, we optimized the monodispersity of our alginate droplets.

In our initial attempts to create alginate droplets the size within a batch often varied significantly. Additionally, due to aggregation a lot of droplets were lost. Optimization of parameters such as flow rate, alginate concentration and N2 pressure led to an increase of monodispersity for all tested sizes (50-300 μm). Specifically, an alginate concentration of 1.8 % alginate and 0.2 % low-viscosity alginate proved to be ideal. Pressure and flow rate determine the droplet sizes.

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Monodispersity of alginate droplets with three different diameters before and after optimization of the hardware device.

Bacteriophages Encapsulated In Alginate Can Withstand Gastric Acid

The main problem of oral application is the acidic environment in the gastric fluid, necessitating protective measures against degradation. The other requirement of phage protection is the release of functional phages in the intestines. For this reason we compared the behavior of the encapsulated phages and non-encapsulated phages in simulated gastric fluid (SGF) and simulated intenstinal fluid (SIF).

In SGF, the number of active non-encapsulated phages decreases by more than 99.99 % within an hour. This shows the urgent need of a form of protection against degradation to make oral application of bacteriophages possible. As a reference, we used phages that were chemically released by citrate from alginate droplets.

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Non-encapsulated phages are highly degraded within an hour in simulated gastric fluid.

In comparison, the encapsulated phages were tested in SGF for the same time as the non-encapsulated phages. Afterwards, the same droplets were exposed for two hours to simulated intestinal fluid to test the release of functional bacteriophages in this environment.

The encapsulated phages were barely released in an hour of exposure to SGF. After transfering the capsules to SIF the number of active phages reached that of the undegraded reference. This indicates that the encapsulation of bacteriophages in alginate capsules enables the possibility of an oral application. Further experiments could test the alginate capsules in an animal model system.

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A small amount of encapsulated phages are released in SGF. In SIF, functional phages are released in high concentration.



Phactory manufactured bacteriophages exhibit the purity requirements for oral application but a higher degree of purity is necessary for intravenous delivery. An increase in purity can be achieved with expensive high performance liquid chromatography. For our manufacturing approach we developed another affordable and easy to use device, the Phactory-Purifier.

This apparatus requires only choosing standard parts, a 3D printed chassis, and is compatible with a custom filter material.

Overall design

Sample purification is achieved by column purification (Biorad Cell 6 column) similar to a HPLC setup. Using phage purifier, the sample can be pushed through the filter by applying pressure to the column. To increase phage purity we used a hydroxyapatite filter. The column with attached filter is fitted in the center of a custom 3D printed cavity. Standard PTFE tubing attached by a tube fitting to seal the cavity allows to fill in the sample with a syringe pump.

Sealing the cavity airtight and waterproof is of high importance to ensure constant pressure within the cavity. The sample has to leave the cavity through the column maintaining a constant flow rate. The extruded sample is fractionized and can be collected in several tubes.

Individual Parts

Column with Hydroxyapatit Filter

Main part of the Phactory-Purifier is the Biorad Cell 6 column completed by a hydroxyapatite filter. The complete unit allows for purification of samples with xx by applying continuous pressure to the sealed system.

3D Printed Cavity

The custom 3D printed device is depicted in Figure XI. As material we chose again PMMA. The cavity has an inlet fitted to the diameter of a standard tube fitting. The outlet is tailored for the tight insertion of a Biorad Cell 6 column. Inlet and outlet have to be sealed airtight and waterproof to allow for the application of continuous pressure within the printed cavity.


  • Three-way cock (Romed STOPCOCK 3-WAY $0.40 per piece)
  • Fitting Elveflow
  • Biorad Column
  • Hydroxapatite
  • Syringe (20ml & 1-3ml Braun Omnifix)
  • Dispensing Tip (ID 0.51mm, Vieweg)
  • Syringe Pump (igem 2016)
  • Pneumatic PTFE Tubing ID 0.5mm
  • 4x M3x20 Screws + Nuts
  • 3D Print of Nozzle (PMMA)
  • Microcentrifuge tubes (15-30 pcs.)


Mask Design

The microfluidic channels were planned in AutoCAD (Figures below), and the design is explained here

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CAD design of fotolithographical mask
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CAD Design of Fotolithographical Mask

Chip Fabrication

Master molds were manufactured via two-step photolithography and microfuidic chips were made from PDMS using soft-lithography, as described in the protocols under the point "cloning".

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Processed wafer as master mold for the microfluidics
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PDMS chip with micofluidics channel imprint


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Comparison of OraColi to other players.

With OraColi we were able to introduce basic population dynamics to an audience of over 1100 people by social media posts with 22 videos. These included predictions to the actual soccer matches of the World Soccer Championship in Russia and explanatory videos about the biological principles behind. For the Human Practice aspect we found OraColi as an easy way to arouse interest in a broad audience for synthetic biology. To measure the ‘predictive power’ of OraColi, we compared OraColi to random predictions and to the predictions made by the bookmakers. Statistical analysis reveals that our bacteria can clearly not predict the outcome of football matches as we had a chance of 1/3 to predict the correct result. As a resume we can say, we were successful in the aspects of education and implementation only while we will never be able to beat professional soccer analysts with OraColi.