Portable Dual Port Filter

Overview

    In Wet Lab and Mathematical Model, we need to characterize the period of oscillator whose property is represented by oscillation of fluorescent signal from reporter GFP. For characterizing population oscillation and standardizing the measurement, we need to make the bacteria synchronized at the beginning, which makes the measurement more meaningful and repeatable. Since the oscillator contains cooperation among three pairs of repressors and promoters and LacI is one of them, we plan to use high concentration of lactose or IPTG to constantly bind LacI and activate pLac, which will express TetR to repress pTetR, which will be repressed to express another repressor cI. Thus, after high concentration of lactose or IPTG treatment, oscillator will cease at LacI+TetR-cI-.
     To realize this target, we need to use some particular solution to treat bacteria in a long enough period before usage but not overlong, so that they can be activated at the same time and then separated for the next experiment. In order to achieve such an experimental goal, we designed our own instruments Portable Dual Port Filter to make such activation procedure easier to operate, and we have verified its effectiveness. This will facilitate later experiments.

Design drawings

Portable Dual Port Filter

1,2: Double pass valve
4: catheter
5: Three-way valve
6,8: Pressure-Controlled needle (including pressure-controlled needle piston)
7: container
3: lid (The structure is as follows)

    Fill the No. 6 syringe with a Bacterial solution, and then fill the No. 2 syringe with water. Press the Bacterial solution into the container. After filtering through the bacteria filiter, the liquid flows out of No. 1 double pass valve, while the bacteria will be trapped by the filter screen. Then use distilled water to flush the filter screen to flush the bacteria out of No. 2 double pass valve. The bacteria-medium (solid-liquid) separation can be realized.

3D Print

    The following is our 3D printing model display.

Experimental Process

Preparation

    1. Prepare and autoclave normal saline solution and 1M disaccharide solution.
    2. Incubate the bacteria in 5mL LB medium with 100 μg/mL Amp at 37℃ for 14h.
    3. Sterilize the model overnight with ultraviolet light in the clean bench.

Consideration: For hardware instrument functional verification, we planned to use Escherichia coli DH5a to represent our engineered bacteria and sucrose to represent lactose due to lack of purchasing time and cost.

Test

    1. Fix a piece of bacteria filter membrane with a pore size of 0.22um into the model in the clean bench.
     2. Split the above bacterial solution cultured overnight, 2mL in an EP tube and 2ml in a 5mL syringe. Keep the EP tube at 4℃ to inhibit bacteria’s growth, which is set as the control group.
     3. Inject 2mL culture into our model with the syringe. Then open the No.1 valve and let it flow out of the lower pipe through the filter membrane.
    4. Inject enough 1M disaccharide solution with another syringe to cover the surface of the filter membrane. After 30min, open the valve and let it flow out of the lower pipe through the filter membrane. Close the valve.
    5. Inject normal saline solution with another syringe to wash the bacteria off the filter membrane. Open another valve and collect the washing liquor into some EP tubes through the pipe on the side of our model. These are the treatment group.
    6. Centrifuge these tubes along with the EP tube at 4℃ for 10min at 3000 rpm.
    7. Remove supernatant. Resuspend the bacteria pellets in 5mL LB medium with 100 μg/mL Amp and incubate at 37℃ for at least 2h. The resuspended bacteria of the treatment group are collected into one test tube, and that of the control group into another.
    8. Measure OD600 with a spectrophotometer and LB medium with 100 μg/mL is used as control. Record the data.
    9. Clean the model with ddH2O and sterilize it with UV for the next experiment.
    10. Repeat the above procedures.

The results are as follows.

    After two independent test experiments, we found the elution efficiency after adherence was very low, for OD600 in treatment group was much lower than that in control group which means that most bacteria were trapped in filter membrane. Thus, we made an attempt to improve it. In the next two experiments, we tried to rinse the used filter membrane into the LB medium before incubation. The results are as follows.

      To our surprise, this simple step improved the results by an order of magnitude. After rinsing, eluted bacteria are much more compared with direct elution . Inspired by the results, we plan to improve our model by designing a mobile elution part on it in the future, in an attempt to increase the elution efficiency and benefit our users.
     During the experiment, we found the filtered liquid was still turbid, so we explored the reason by comparing the filter membrane we used with the commercial filter membrane. The latter turned out to be clear as expected. Given that we only used one piece of filter membrane while the commercial one consists of multiple layers, we may make some improvements and find a better way to fix multiple filter membranes in our model in the future.

Figure Comparison of elution efficiency between Direct Elution and Rinse + Direct Elution Group.

Summary

     In summary, we successfully achieved a portable dual port filter for synchronization of bacteria in wet lab and an enteral drug simulation equipment to explore the best combination of capsule construction. In the future, we will consider more details to make the model closer to the real application. 3D printing plays an integral role in manufacturing equipment, and we hope to adopt bio-3D printing technology to obtain a more realistic intestinal environment in the future.

Enteral Drug Simulation Equipment

Overview

     In our project, our engineered bacteria are supposed to habitat in human intestine, so we need some drug delivery system to release them in target intestine rather than stomach with extreme low pH which will absolutely kill engineered bacteria. So we came up with an idea to use a kind of capsule.
    In hardware, to characterize the breakdown dynamics of drug capsules as they pass through the human digestive track, we designed a device to simulate the track and the environment within it. This will help to explore the appropriate size and material of drug particles, so that they can pass through the mouth, esophagus and stomach, and then gradually dissolve after reaching the intestinal tract, and finally bacteria are allowed to colonize the intestinal tract. Our device takes into account more details, including the peristalsis of the intestine and the flow of fluid, which will make our results more credible.

Capsule Design

     Finally, we designed such multi-layer capsules, within which engineered bacteria ceased by lactose are enclosed. In the outermost layer we used gelatin, which slowly dissolves in any pH solution. This prevents the particles inside from being melted directly in the mouth, thus helping the particles enter the stomach. The outer layer of the capsule stays in the stomach for two hours and then ablates, exposing the particles inside and entering the intestine. The outer layer of the pellet is wrapped in carboxymethyl cellulose and is not dissolved in an acidic environment, but is dissolved in a neutral environment. This will ensure that the outermost layer of the granules is not digested in the stomach and can be smoothly absorbed into the intestines. After entering the intestine, the carboxymethyl cellulose of the outer layer of the granules falls off, exposing the engineered bacteria and lactose to the outside The lactose is dissolved with the intestinal fluid, forming a local high-concentration lactose solution and synchronize the engineered bacteria. Then engineered bacteria are expected to colonize the intestines and exert their curative effects.

    Figure Capsule Concept Graph

Equipment

    In order to more realistically simulate the situation of human intestine, we need to repeatedly squeeze the coarse catheter and let the solution in the coarse catheter flow.

    Finally, After a series of production, our equipment is as follows:

    1: Microflow pump
    2: Synchronous engine and rotor
    3: Coarse catheter
    4: Frame
    5: A thick conduit pipe
    6:Joint

3D Print

    The following is our 3D printing model display.

Experimental Process

Pilot Experiment

    1. Use molds to make particles of various substances(Carboxymethyl cellulose, Carbomer, Sodium alginate) of the same radii(Diameter is 0.5cm).
     2. Place in aqueous solution of ph=5.6 and hydrochloric acid solution of ph=1.85, soak for 2h, and observe the particle.

Figure Mold for particles and particle products with different radii

     In order to make particles with the same radius, we bought a mold. Mix the powder particles with the same amount of water, knead them into a ball, and put them into the mold. Bake in the mold for shaping, and finally form equal sized particles.

    annotation: Invert the tube. On the left is the result of carboxymethyl cellulose soaking in water for two hours, and on the right is the result of carboxymethyl cellulose soaking in hydrochloric acid for two hours. It can be found that carboxymethyl cellulose in hydrochloric acid will not dissolve, but will settle in a gel-like status at the bottom.

Formal Experimentation

    1. Prepare hydrochloric acid solution with pH=1.73 to simulate the ph value of stomach.

    annotation: Configure hydrochloric acid using a pH meter measurement

    2. Use the mold to make particles with different radius(Diameter is 0.3cm, 0.5cm, 0.7cm).
    3. Put particles in the Coarse catheter. Set up the instrument.
    4. Fill the middle coarse catheter with solution until it passes through the outlet and inlet.
    5. Turn on the Synchronous engine. Turn on the microflow pump.
    6. Stand it for two hours and observe whether the particles dissolve.
    7. Change hydrochloric acid into water and repeat the experiment.

    This is the change of particles under hydrochloric acid immersion. It can be found that particles of any size will not dissolve in acid solution.

    We found that the particles of carboxymethyl cellulose with radius of 0.3cm had been dissolved in water, but not dissolved in acid solution. This shows that this substance can be used as a wrap outside the particles. The granular material was made of carboxymethyl cellulose and the radius should be less than 0.3cm to ensure that it completely dissolves in the intestine and can stay in the stomach for at least 2 hours.

Summary

    In summary, we achieved an enteral drug simulation equipment. In the future, we will consider more details to make the model closer to the real gut environment. 3D printing plays an integral role in manufacturing equipment, and we hope to adopt bio-3D printing technology to obtain a more realistic intestinal environment in the future.

A Bill of Purchasable Materials

Portable Dual Port Filter

Enteral Drug Simulation Equipment

Reference

[1]Elowitz, M., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000). https://doi.org/10.1038/35002125
[2] Bowtle W J. Materials, process, and manufacturing considerations for lipid-based hard-capsule formats[M]//Oral Lipid-Based Formulations. CRC Press, 2007: 101-128.
[3]Duke G E. Alimentary canal: secretion and digestion, special digestive functions, and absorption[M]//Avian physiology. Springer, New York, NY, 1986: 289-302.