Team:Lambert GA/Hardware

HARDWARE

OVERVIEW

Lyophilization is a method of freeze-drying bacterial samples increasing the sample’s shelf life and biosafety. Cells enter a hibernated state as the ice is sublimated outside the sample, leaving a pellet of freeze-dried cell content [1]. After being lyophilized, bacterial biosensors do not need to be held in refrigerated containers. Additionally, there is a reduced chance of denaturing during transit and storage. Commercial lyophilizers range from $8,000-14,000, making them cost-prohibitive to underfunded labs across the world (Specific model: Labconco Modulyo-D )[2]. LyphoX, a frugal vacuumed powered lyophilizer, reduces costs by 99% and creates a mobile alternative that can be utilized in remote locations lacking access to consistent refrigeration. Bacterial biosensors and cell-free lysates were developed and used to test LyphoX. The LyphoX committee conducted numerous tests with samples at an optical density (OD600) of 1.0. in order to establish a protocol. After analyzing the data, a vacuum cycle of 90 minutes, reaching pressures of 600 PA, and a simultaneous chilling process in a 0°C unit for 90 minutes produced optimal results. Allowing ice in the samples to sublimate produces freeze-dried cells and cell parts, thus reducing denaturing of samples. Once lyophilized, the cells can hold a consistent shelf life of at least 6 days in a room-temperature environment. The cells can then be rehydrated and used for sensing. Partnerships with Florida State University and Johns Hopkins are ongoing, testing the efficacy of LyphoX equipment and protocols. At $109, LyphoX offers a mobile, cost-efficient alternative to commercial lyophilizers, making the transportation of cells accessible to a wider range of labs across the globe.

Figure 1. Water Phase Diagram. Sublimation region is the red-dashed region in the Vapor portion.

PARTS

  • Electric Vacuum Pump (110 V) 4 CFM - 5 Pa - ⅓ Hp - $45
  • Metal Container - $10
  • Plastic Container (that can comfortably fit the metal container inside) - $5
  • Analog Pressure Gauge - $7
  • ⅜ Reinforced Tubing - $5 (40¢ /ft.)
  • Ice - $0
  • Rubber Sheets for Gasket - $5
  • Male Hose Barb Adapter ⅜ in. (3) - $12
  • Female Hose Barb Adapter ⅜ x ¼ in. (1) - $4
  • 8 in. x 10 in. Lexan Clear Sheet - $6
  • ½ to ¼ in. Hose Clamp (2) - $2
  • Plumbers Tape - $2
  • Plumbers Putty - $4
  • Vaseline - $2

Total: $109

ASSEMBLY

Figure 2. LyphoX running vacuum cycle cell samples.


Figure 3. LyphoX schematic with labeled compartments.

KEY FOR ASSEMBLY DIAGRAM

  1. 16-inch segment of tubing
  2. Female Hose Barb Adapter ⅜ x ¼ in
  3. ½ inch hole
  4. ⅜ inch hole
  5. Male Hose Barb Adapter ⅜ in.
  6. Rubber Gasket

INSTRUCTIONS FOR ASSEMBLY

  1. Cut the reinforced tubing into a 16-inch segment (1).
  2. Attach the segment of reinforced tubing (1) to the vacuum pump by screwing one female hose barb adapter (2) to the outlet of the pump.
  3. Drill a 1.27 cm (½ inch) hole (3) and a .9525 cm (⅜ inch) hole (4) near the center of the Lexan sheet with a 6 cm (2.36 inch) space between the two holes.
  4. Screw the pressure gauge into the smaller hole (4).
  5. Screw the other barb adapter (5) into the Lexan sheet (3). Seal it with a hose clamp.
  6. Cut a circle in the center of the rubber gasket (6) ½ inches smaller than the diameter of your metal container.
  7. Apply Vaseline to any area that comes into contact with the gasket.
  8. Use Plumber’s Putty, Plumbers Tape, and hose clamps to seal any brass or tube interactions.

DIRECTIONS FOR USE

With extensive testing, we established a standardized protocol including cell preparation and trial length that may be replicated by other teams.

  1. Fill a row of 4 PCR tubes with 30 μL of the desired cell culture in each tube. Place tubes in the freezer until completely frozen. Poke holes in the caps with a metal point.
  2. Fill the plastic container halfway with ice water and place the metal container inside. Place all of this in the freezer until fully frozen.
  3. Using a wipe of some sort, wipe condensation from the inside of the container until dry.
  4. Tape PCR tubes to the inner wall of a circular metal container.
  5. Place the gasket on top of the metal container, making sure that it fully covers the edges of the metal container.
  6. Place the Lexan sheet lid on top of the gasket. Make sure the valve suction point is not directly on top of the PCR tubes.
  7. Place the whole system in an insulated container (styrofoam).
  8. Turn on the pump and run for 90 minutes.
  9. Take the PCR tubes out of the metal container and leave them at room temperature until needed. Replace the lid of the PCR tubes with ones without holes.

RESULTS

DATA (LyphoX)

Trial Number Initial Weight (g) Final Weight (g) % Water Sublimated
1 0.767 0.650 97.5%
2 0.766 0.646 100%
3 0.770 0.663 89.2%
4 0.766 0.656 91.7%
5 0.766 0.650 96.7%
6 0.766 0.651 95.8%
7 0.770 0.650 100%
8 0.767 0.649 98.3%
9 0.773 0.653 100%
10 0.768 0.650 98.3%

Table 1. Table of data collected from LyphoX tests. *Percent water sublimated was calculated by dividing the weight change by 0.120 (30μL x 4 = 120μL = .120 grams)

DATA (Labconco Modulyo-D)

Trial Number Initial Weight (g) Final Weight (g) % Water Sublimated
1 0.744 0.624 100%
2 0.737 0.618 99.2%
3 0.748 0.629 99.2%
4 0.740 0.623 97.5%
5 0.746 0.629 97.5%

Table 2. Table of data collected from commercial lyophilizer Labconco Modulyo-D tests. *Percent water sublimated was calculated by dividing the weight change by 0.120 (30μL x 4 = 120μL = .120 grams)


NEGATIVE CONTROL


Our team decided to utilize a negative control group in order to identify the direct effect of lyophilization on frozen cell samples. Bacterial specimens, including E. coli, are susceptible to damage caused by the freeze-thaw process [3]. To combat this, cells are typically stored in a cryoprotectant, specifically a glycerol stock, when placed inside of a freezer in order to protect them from membrane damage and DNA mutation [4].

In both negative control and experimental groups, cells were stored and frozen without glycerol and left at room temperature. The only difference between the two groups was the presence of lyophilization: the experimental group was freeze-dried with our frugal lyophilizer before being left at room temperature.

After leaving both the experimental group and the negative control at room temperature for a day, we rehydrated and plated the resulting cell matter. The negative control exhibited no growth of rehydrated cells (See Fig. 11), while the experimental group showed adequate cell growth after up to six days of being left at room temperature (See Fig. 4-10).

The results of this experiment illustrate that LyphoX is effective in preserving the cellular structure of bacterial samples and that longer-term storage at room temperature is viable through lyophilization even in the absence of a cryoprotectant.

Figure 11. Rehydration results for the negative control group - frozen then left at room temperature for one day.

Comparing LyphoX to Labconco Modulyo-D proves that our frugal lyophilizer yields similar results to that of a commercial lyophilizer. Even though the Labconco Modulyo-D sublimates a greater average of water weight at 98.68%, LyphoX sublimates an average of 97.58% of water weight using its standardized protocol. This preserves cell samples for several days at room temperature. Additionally, by comparing the rehydration results of lyophilized cells to the negative control group, we can conclude that LyphoX preserves the cellular properties of test samples. Improvements can be made on LyphoX by extending the shelf-life of lyophilized cell samples. By collaborating with faculty at the Bhamla Lab, located at the Georgia Institute of Technology, Lambert iGEM’s hardware committee gained new ideas and improved their project. Specifically, Lambert iGEM’s lyophilizer committee met once a month with faculty members Dr. Bhamla and Mr. Poorna over Zoom to discuss current progress and ways to enhance LyphoX. Under their guidance, we were able to develop new innovative solutions to problems because of their extensive background in biology, physics, and engineering.

Figure 12. LyphoX’s consistent results after completed trial (Table 1. Trial 3).

Figure 13. LyphoX’s consistent results after completed trial (Table 1. Trial 9).

Figure 14. LyphoX’s consistent results after completed trial (Table 1. Trial 10).


APPLICATION


To further validate LyphoX’s proof of concept, we also freeze-dried our phosphate sensor in order to demonstrate that our sensors still function properly after lyophilization (See: Proof of Concept). As shown in Figure 15, we confirmed that our biosensor was expressing GFP both before and after lyophilization, validating that LyphoX did not compromise the samples’ cell structure. Additionally, the successful sequencing results showed that the freeze-drying process did not introduce contamination or damage to the DNA (See Fig. 16).

Figure 15. Visible GFP expression of our phosphate sensor before (left) and after (right) lyophilization.


Figure 16. Sequencing results of our phosphate sensor aligned with the original sequence.

COLLABORATION

GSU

Lambert iGEM also collaborated with the iGEM team at Georgia State University (GSU) and Dr. Matthew Brewer, senior academic professor in biology, to discuss testing for LyphoX and to learn more about both of our iGEM projects. We sent GSU our frugal lyophilizer to validate the replicability and effectiveness of the standardized procedure in freeze-drying cell solution. Georgia State University’s team members visited our lab on October 15, 2021. After running two tests with LyphoX, they delivered promising results. Incubating the cell solutions after four days of storage at room temperature yielded successful growth on plates, validating that users outside of our lab are effectively able to replicate our standardized procedure (see Fig. 17). Additionally, GSU presented us with their iGEM project HNOSS: a biotechnology solution to the growing problem of hair loss. We also presented our iGEM project, Agrosense, to GSU’s team members. Through this collaboration, we were able to validate the effectiveness of LyphoX from a user outside of our lab and explore each other’s iGEM projects.


Figure 17. GSU’s rehydration results after delivering test results to Lambert on 10/15/2021 - both left out for four days at room temperature.


FSU, JHU, UPENN, MIT ALLIANCE


This year, Lambert iGEM entered an alliance with iGEM teams at Florida State University (FSU), Johns Hopkins University (JHU), University of Pennsylvania (UPenn), and Massachusetts Institute of Technology (MIT). Over a period of three months, Lambert iGEM joined a series of virtual meetings to discuss medaling requirements and visualize project goals.

Each team presented their projects and helped brainstorm project ideas and updated their presentations after receiving feedback. In addition, the JHU team created a Minecraft server for the five teams to create visual representations of each project and to form more friendly, personal connections outside of the lab through collaborative entertainment (see Fig. 18). Through this collaboration, we were able to learn about a wide variety of other iGEM projects and, more importantly, establish a partnership specific to Johns Hopkins University and Florida State University in order to address gaps in each of our projects.

Figure 18. Picture of Lambert and Johns Hopkins’ Minecraft project build illustrating their iGEM projects.

PARTNERSHIP

Through the previously mentioned alliance consisting of the 4 iGEM teams, Lambert iGEM was able to organize a partnership specifically with the teams at JHU and FSU to utilize LyphoX, greatly assisting the development of each team’s project goals. After having continuous zoom meetings and GroupMe communication from August 2021 onward, we realized that each team had its respective gaps in its projects. FSU wanted to apply their chitin-secreting cells to another project for validation, and JHU needed chitosan, an antifungal substance derived from chitin, in order to forward their iGEM project regarding coral reef preservation. LyphoX was proposed as a method of making effortless cell transport between JHU and FSU. Without lyophilization, FSU would have to transport an unstable and biohazardous material over a long distance to JHU, creating avoidable costs and time restrictions that impede JHU’s accessibility to an integral aspect of their project. Because of this, all three teams agreed to apply LyphoX to their projects.

FSU first sent their chitin-secreting cells to our lyophilizer committee, who inoculated them in Luria broth and used the frugal lyophilizer to freeze-dry the 0.2mL tube cell samples. After completing the test, our team shipped these samples to JHU; however, due to lab restrictions, JHU is currently unable to acquire petri plates to successfully grow lyophilized cells. Updates are expected before the Judging sessions.

Our partnership with JHU and FSU filled the gaps in each respective project, allowing everyone involved to forward their iGEM project goals. Finally, it was through this collaboration that we further validated our frugal lyophilizer was able to freeze-dry cells for transport across the country.

REFERENCES

[1] Wessman, P., Håkansson, S., Leifer, K., & Rubino, S. (2013, August 3). Formulations for freeze-drying of bacteria and their influence on cell survival. Journal of visualized experiments : JoVE. Retrieved October 20, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846756/.

[2] Labconco. (n.d.). Freeze dryers. Labconco. Retrieved October 20, 2021, from https://www.labconco.com/category/freeze-dry-systems.

[3] Sleight, S. C., Wigginton, N. S., & Lenski, R. E. (2006, December 5). Increased susceptibility to repeated freeze-thaw cycles in escherichia coli following long-term evolution in a benign environment. BMC evolutionary biology. Retrieved October 20, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698501/.

[4] Calcott, P. H., & Gargett, A. M. (1981, February 1). Mutagenicity of freezing and thawing. OUP Academic. Retrieved October 20, 2021, from https://academic.oup.com/femsle/article/10/2/151/610210.