iGEM 2019 — Team BrownStanfordPrinctn

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# Team:BrownStanfordPrinctn/Notebook

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# Diagnostics Notebook

To view detailed notes on the procedures, protocols, etc. used for all work associated with diagnostics, please view the attached PDF document, linked  [here](https://static.igem.org/mediawiki/2019/5/53/T--BrownStanfordPrinctn--PaperMicrofluidicsNotebook1.pdf) and [here](https://static.igem.org/mediawiki/2019/f/f2/T--BrownStanfordPrinctn--PaperMicrofluidicsNotebook2.pdf).

# Drug Production Notebook

To view detailed notes on the procedures, protocols, etc. used for all work associated with drug production, please view the attached document, linked  [here](https://static.igem.org/mediawiki/2019/b/bb/T--BrownStanfordPrinctn--DrugProductionNotebook.pdf).

# Cell-free Notebook

To view detailed notes on the procedures, protocols, etc. used for all work associated with cell free, please view the attached PDF document, linked  [here](https://static.igem.org/mediawiki/2019/8/8d/T--BrownStanfordPrinctn--CellFreeNotebook.pdf).

# Lyophilization Notebook

To view detailed notes on the procedures, protocols, etc. used for all work associated with lyophilization, please view the attached PDF document, linked  [here](https://static.igem.org/mediawiki/2019/f/fa/T--BrownStanfordPrinctn--LyophilizationNotebook.pdf).

# PDMS Microfluidic Device and Expression Systems Notebook

To view detailed notes on the procedures, protocols, etc. used for all work associated with the PDMS microfluidic device and expression systems, please view the attached PDF document, linked  [here](https://static.igem.org/mediawiki/2019/5/51/T--BrownStanfordPrinctn--WillandAlexNotebook.pdf).


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# Team:BrownStanfordPrinctn/Experiments

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The protocols below were implemented by multiple subprojects. All are documented in Benchling notebooks linked in the Notebooks section.

### Designing and assembling constructs: Diagnosis AND Synthesis

**Linearizing pET-23b(+)**

1. Make master mix- USE Q5-- 50 μL recipe:
2. 19 uL qH2O
3. 2.5 μL forward primer (10μM dilution)
4. 2.5 μL reverse primer (10μM dilution)
5. 1 uL Template DNA (10ng plasmid miniprep)
6. 25 uL of Q5 mastermix

**Q5 Thermocycling Procedure:**

1. Initial Denature at 98°C for 30 sec
2. Denature at 98°C for 10 sec
3. Annealing at 61°C for 30 sec
4. used NEB Annealing calculator: https://tmcalculator.neb.com/#!/main, use final concentration of primer in mix (0.5uM)
5. Extension at 72°C for 35 seconds(Q5 is much faster than Taq, and requires 10 sec per kb)
6. Go to step 5 X28 times
7. Final extension at 72°C for 2 min
8. Hold 10°C forever

**Linearizing pBS2E:**

1. Dilute 200uM primer concentration to 10uM
2. ALIQUOT OF PRIMER: 2ul of primer + 38ul of water ==> 40ul of 10uM
3. Make master mix- USE Q5-- 50 μL recipe:
4. 25 ul Q5 master mix
5. 2.5 μL forward primer (10μM dilution)
6. 2.5 μL reverse primer (10μM dilution)
7. 1uL Template DNA (a couple nanograms worth)
8. 19uL of aliquoted qH2O

**Q5 Thermocycling Procedure:**

1. Initial Denature at 98°C for 30 sec
2. Denature at 98°C for 10 sec
3. Annealing in a temperature gradient of 72˚C for 30 sec
4. used NEB Annealing calculator: https://tmcalculator.neb.com/#!/main
5. use final concentration of primer in mix (not 10uM, actually 0.5uM bc added 1.25 uL of 10uM into a total volume of 25uL)
6. Extension at 72°C for 155 seconds
7. Q5 is much faster than Taq, and requires 20-30 sec per kb.
8. 6.2 kb x 25 seconds = 155 seconds
9. Cycles: X28 cycles
10. Final extension at 72°C for 2 min
11. Hold 10°C forever

**PCRing overhangs and his tag onto hG-CSF and teriparatide:**

Dilute 200uM primer concentration to 10uM. Using the 10 uM dilution of the primer in the mix.

**Make master mix- USE Q5-- 25 μL recipe:**

1. 9.5uL qH2O
2. 1.25 μL forward primer (10μM dilution)
3. 1.25 μL reverse primer (10μM dilution)
4. 0.5uL Template DNA (using originally ordered inserts at 10 ng/uL.)
5. 12.5 uL Q5 2X Master Mix

**Q5 Thermocycling Procedure:**

1. Initial Denature at 98°C for 30 sec
2. Denature at 98°C for 10 sec
3. Annealing at 57°C (teriparatide) and 69°C (hG-CSF) for 30 sec
4. used NEB Annealing calculator: https://tmcalculator.neb.com/#!/main
5. use final concentration of primer in mix (not 10uM, actually 0.5uM bc added 1.25 uL of 10uM into a total volume of 25uL)
6. Extension at 72°C for 15 seconds(Q5 is much faster than Taq, and requires 10 sec per kb.)
7. Go to step 5 x28 times
8. Final extension at 72°C for 2 min
9. Hold 10°C forever

**Gibson Assembly**

1. Preheat a thermal block to 50C.
2. Final amount of vector should be 50-100 ng. Divide 100 ng by the vector concentration from Nanodrop to get the volume of vector you should add.
3. Use https://nebiocalculator.neb.com/#!/ligation to calculate the amount of insert you need. Make sure to put the correct bp lengths for both the vector and the insert. The calculator will give you a result in ng.
4. Take the nanogram result from the calculator and divide by the insert concentration (from Nanodrop) to get the volume of insert you should add.
5. If the volume is less than 0.50 uL, make a 1:10 dilution and add 10 times the volume.
6. Example: we would need to add 0.32 uL of insert. Take 1 uL of the insert and add to 9 uL of H20. In the final tube, you would then add 3.20 uL of the dilution.
7. Make a table with your samples and the components: Vector, Insert, H20, 2x MM.
8. The vector volume is calculated from Step 2, and the insert volume is calculated from Step 3. If you are using a dilution, make sure to put the volume of the dilution and not the original volume.
9. The 2x MM volume is set at 10 uL.
10. The final volume of all tubes should be 20 uL. Using the other three known volumes, subtract from 20 uL to find the volume of H20 needed.
11. All components should be on ice, vortexed, and centrifuged. Add them to PCR tubes. Vortex and centrifuge the tubes.
12. Place tubes on thermal block and incubate for 15 minutes (if doing 2 fragments).
13. If 4 or more fragments, do 1 hour incubation.
14. Following incubation, store samples on ice or at -20C for subsequent transformation.

**Transformation**

1. Allocate 25ul of NEB 5-alpha or T7 competent cells to centrifuge tubes on ice for 10 minutes.
2. Add 1ul of chilled assembled product to the centrifuge tubes.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock for 30 seconds (5-alpha cells)/10 seconds (T7 cells)
5. Place on ice for 5 minutes. Do not mix.
6. Pipet 950ul of room temperature SOC into the mixture.
7. Incubate at 37˚C for 60 minutes and shake at 250rpm.
8. Warm selection plates with appropriate antibiotics at 37˚C.
9. Before plating, pipet SOC media and cells up and down to mix evenly.
10. Plate 50ul, incubate at 37˚C for 12-16 hours.

**Protocol for SDS-Page Gels:**

**Using ThermoFisher Bolt Bis-Tris Plus Mini Gels:**

1. Add loading buffer (2x concentration, in 20ul of sample, add 20ul of loading buffer)- Incubate at 70˚C for 10 min- After 10 min cool samples down on the table and spin to get rid of evaporate from the cup- Rinse gel with DI water after tearing off the foil- Tear off the white strip on the bottom- Green line shows what level to fill up with loading buffer before putting gel in (0.5 L for 1 gel - Bolt MES SDS running buffer: https://www.thermofisher.com/order/catalog/product/B0002)- Pull grey handle to secure the gel make sure the electrodes are positioned on the inside to match lid- Rinse wells with 100ul running buffer- Run at 150 V for 30-40 min- Prepare 100 ml acetic acid 7.5% (100% acetic acid is under the hood) (10 ul aliquots + 50 ml acetic acid (7.5%))- Stop gel. Rinse with DI before opening- Crack the gel open.- Push the gel through the opening where the white tape was- Stain for 30 min (protect from light). Cyber-orange binds to SDS envelope around protein- Shake gently.- Rinse with diH2O- Wash 15min (up to 1h) with acetic acid without stain- Rinse 30 sec with DI H2O just before using the scanner

Scan:
Filter 526 (or 520 for stronger signal less acuity)
excitation 488 nm

### Inducing Protein Synthesis: Diagnosis AND Synthesis

**Overnight induction with IPTG**

1. Resuspend a single colony in 5ml LB with antibiotic in a 15mL Falcon tube. Let grow overnight at 37o C in a shaking incubator.- Prepare a 1000ml conical flask with 0.5L of LB + antibiotic.- Inoculate flask with 10ml culture of starter culture.- Incubate at 37o C until OD600 reaches 0.4 - 0.6.- Be sure to take uninoculated LB + antibiotic to use as a starting blank.- Measure first OD just after inoculating.- Measure every 0.5 hour until OD reaches 0.2, then check every 15 mins until OD range is attained.- Add IPTG to a final concentration of 0.4mM.- Induce for 2 hours in a shaking incubator at 37o C.- Spin down until a pellet is observed.- Pour off supernatant, and flash freeze at -80o C overnight.

**Lysing Using B-PER Reagent:**

1. Pellet bacterial cells by centrifugation at 5,000 x g for 10 mins- Add 4 ml of Thermo Fisher B-PER Reagent per gram of cell pellet. Pipette the suspension up and down until it is homogenous- Incubate 10 - 15mins.- Spin down until pellet is visible. The pellet will contain cell membrane, cell structure and any inclusion bodies that have formed. The supernatant is cell lysate. Save both fractions until it is confirmed that your protein of interest is in the lysate fraction.- Store at 20C.

### DIAGNOSIS: Printing with Experimental Lysate:

**Suggested printers:**

Our team worked with the Canon Pixma MP950 and the Canon Pixma TR4520 to print our proteins. While the MP950 is in general a higher end Canon printer, for this particular purpose it was not well suited. Error messages that halted function (that are apparently systemic to all units) were inhibitory when we were trying to make the printer “forget” about tampered parts of its hardware. Also the ink cartridges had reader chips that most likely contained information about the color in the cartridge, and whether it had been used before. For a project that is looking to tamper with cartridges, this was not an ideal characteristic.

The TR4520, on the other hand, worked extremely well for our needs. It held only two cartridges, color (containing compartments with blue, red, and yellow) ink and black ink, which made it extremely easy to control exactly what was being printed. For example, we left the color cartridge intact, and in our print surrounded where we wanted our protein to be (signified with black ink on our printing template) with a bold blue background. This eased visualization of where our protein should have been printed, and also provided a helpful negative control when our testing liquid ran past its final destination in our microfluidic device. This printer was also extremely economical and easy to attain.

**Prepping Cartridge for Lysate Loading:**

We decided to commandeer the black ink cartridge for our experimental lysate. This is how we did it:

1. Locate the lid, and the seam that connects it to the body of the cartridge.- Using a large screwdriver, press into this seam until the cartridge pops off, or can be torn off. Try to make this as clean a separation as possible, as the lid needs to be reattached for adequate loading into the printer.- Remove the white sponge from the cartridge, and clean with DIwater until runoff is clear when sponge is squeezed.- Rinse inside of cartridge and printhead with DIwater. The printhead is the interface between the sponge and the paper to be printed on. Rinse until runoff from all directions is clear.- Let both components dry/ dry off cartridge with paper towel.

**Loading Experimental Lysate:**

1. Pipette ~1ml of experimental lysate onto printhead pad embedded on the inside of the cartridge. This will aid flow of protein through printhead.- Pipette ~2ml of experimental lysate onto depressed square shape on white sponge, making sure it’s well saturated.- Pop sponge back into the cartridge, making sure the depressed square on the sponge itself is aligned with the printhead pad.- Put lid back on cartridge, using scotch tape to affix it to the body.- Load cartridge back into the printer.- Go forth and print.

### SYNTHESIS: CELL-FREE SYSTEM

**A detailed and formatted guide to cell-free system production can be found linked [here](https://static.igem.org/mediawiki/2019/6/68/T--BrownStanfordPrinctn--cellfreeproto.pdf)**

### SYNTHESIS: CELLULAR LYOPHILIZATION

1. Culture cells overnight in its proper broth medium (E. coli, LB; B. subtilis, LB; Vmax, 2xYTP) supplemented with proper antibiotic- Next day, spin down cells in centrifuge. Remove supernatant.- Resuspend in lyophilization media (dilute cells 1:40)- Prepare 96 well-plate with samples by aliquoting 100uL of sample into each well- Set-up lyophilizer, and lyophilize overnight (ideally 12-48 hrs, depending). Remember to freeze cells beforehand.- After lyophilization is complete, rehydrate time point t=0.- Sterilize an 96-well plate and reagent reservoir.- Retrieve proper broth medium and 0.9% saline solution from fridge.- Fill reservoir with saline solution, and pipette 90uL into five rows of the 96-well plate- Fill one row of lyophilized 96 well-plate with 100uL of broth medium- Use a multichannel pipette and remove 10uL of broth medium into pipette into the first row of the 96 well-plate; mix thoroughly.- Take 10uL of solution from the first row of the 96 well-plate and release the solution into the second row of the 96 well plate- Repeat f. for all five rows - serial dilution is performed.- Prepare a large agar plate for bacteria culture.- Using the multichannel, pipette 10uL from the 5th row of the 96-well plate (this is the most diluted sample), and release the solution near the side of the culture plate- Then pipette 10uL from the 4th row of the 96 well-plate, and release the solution on the agar plate slightly above the 5th row. Repeat for all remaining rows.- Take 10uL from the rehydrated sample on the sample 96 well plate and pipette onto the plate as well. Make sure it is above the least diluted sample row.- Let the samples dry on the plate.- Label plate and seal it with parafilm- Let incubate at 37C overnight.- Next day, take photos and count cells.- Calculate cell viability.

### PURIFICATION: EXPRESSION CHIP MANUFACTURE

**SLA 3D printing (1st iteration)**

1. Upload SolidWorks STL file into SLA formlabs “Form 2 Desktop SLA 3D printer”- Use Preform Slicing software to adjust printing parameters, 25 micron layers, create suitable support structures (verified by Preform), select resin type (Formlabs clear resin).- Begin SLA printing, 10-15 hours- Remove structure from printer- Place in isopropyl bath, 20 minutes- Remove structure from isopropyl bath- Clean excess resin/isopropyl off with alcohol wipes/paper towel- Place in UV hardener, 6-8 hours- Remove supports with pliers- Use sandpaper to smooth outer surfaces- Use needle to carefully outline the inner faces of the two structures with uncured resin- Press two halves of the structure together, using a vice- Use UV lamp to cure the two halves together, 12-20 hours

**SLA 3D printing (2nd iteration)**

1. Upload SolidWorks STL file into SLA formlabs “Form 2 Desktop SLA 3D printer”- Use Preform Slicing software to adjust printing parameters, 100 micron layers, create suitable support structures (verified by the Preform), select resin type (dental resin).- Begin SLA printing, 10-15 hours- Remove structure from printer- Place in isopropyl bath, 20 minutes- Remove structure from isopropyl bath- Clean excess resin/isopropyl off with alcohol wipes/paper towel- Flow isopropyl alcohol through chip with a syringe, clearing uncured resin out of the channels- Place in UV hardener, 6-8 hours- Remove supports with pliers- Use sandpaper to smooth outer surfaces

### Interfacing

1. Cut four 1-2 inch lengths of PEEK tubing, 1/8 inch diameter- Use sander to flatten the interfacing side- Coat the end of the tube in epoxy (for each tube)- Hold against the interface of the expression chip until hardened, 5 minutes- Attach latex tubing to the ends of each PEEK tubing interface

### PURIFICATION: PDMS DEVICE MANUFACTURE

**Photolithography**

**Wafer Preparation**

1. Dehydration bake wafer on a hot plate at 115°C for at least 3 minutes- Rinse wafer in methanol- Blow dry with nitrogen gas- Bake at 95°C for 10 minutes to dry solvent

**SU-8-2005 Spinning**

1. Pour SU-8-2005 on wafer, covering at least ⅔ of surface- Spread by spinning 500 rpm, 10 s, 133 rpm/s acceleration- Cast by spinning 3000 rpm, 40 s, 266 rpm/s acceleration- Soft bake on a hot plate at 65°C for 2 minutes, 95°C for 3 minutes, 65°C for 2 minutes- Expose for 20 seconds at 8.4 mW/cm2 (124 mJ/cm^2) with no mask- Bake on a hot plate at 65°C for 2 minutes, 95°C for 4 minutes, 65°C for 2 minutes

**Flow Layer**

1. Pour SU-8-2075 generously, spinning bottle while pouring to avoid bubbles and streaking- Spread by spinning 500 rpm, 10 s, 133 rpm/s acceleration- Cast by spinning 1300 rpm, 33 s, 266 rpm/s acceleration- Let sit on flat surface for at least 20 minutes to allow photoresist relaxation- Soft bake on a hot plate at 65°C for 5 min, 95°C for 30 min, 65°C for 5 min- Expose for 65s at 230 mJ/cm^2, split into two exposures separated by 90s, each for 32.5s, place mask over wafer for the exposure- Hard bake on a hot plate at 65°C for 5 min, 95°C for 12 min, 65°C for 5 min- Submerge in SU-8 developer for 10-13 minutes, until the design becomes visible on the wafer- Hard bake from 65-165°C for 2 hours at 120°C/hour

**PFOS Treatment of Wafer**

1. Pour 1-2 drops on PFOS into bottle cap, place cap in bell jar- Place wafers in bell jar next to cap, seal and pull vacuum into bell jar for 1 minute- Switch to no pull to stabilize bell jar, let PFOS deposit for 10 minutes before depressurizing- Place wafers in clean petri dish- Pour 20g of 1:10 thinky mixed PDMS onto each wafer, place in over at 80°C for 1 hour and remove PDMS layer

**PDMS Fabrication**

1. Blow dry cup with clean air to remove dust, pour Part A and Part B in a 10:1 ratio of A:B with at least 40g of PDMS for each wafer- Mix for 3 minutes in THINKY mixer at 2000rpm and defoam for 3 minutes- Place wafer in aluminum foil wafer holder which wraps around base of wafer and has walls at least 1” tall- Pour 40-45g PDMS on each wafer, place in vacuum chamber and degas for about 45 minutes- Break vacuum briefly if bubbles in PDMS look like they will rise out of wafer holder, then re vacuum- Bake at 80°C for 60 minutes in convection oven or hot plate- Peel PDMS off of wafer, cut devices out using scalpel and cut inlets and outlets with hole punch, make sure to remove PDMS plugs from punched holes

**Plasma treatment**

1. Blow dry glass slide and PDMS layer to remove dust- O2 plasma treat glass and PDMS at 150W for 4.5 minutes in air using a PDC-001 Air Plasma Cleaner- Place device on glass substrate and bake for 24 hours

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# Team:BrownStanfordPrinctn/Results

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[Part Collection](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Hardware)
[Measurement](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Measurement)
[Model](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Model)
[Plant](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Plant)
[Software](https://2019.igem.org/Team%3ABrownStanfordPrinctn/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=BrownStanfordPrinctn)

![](https://static.igem.org/mediawiki/2019/8/8c/T--BrownStanfordPrinctn--Astronaut_Results.jpeg)

### Diagnostics: Fusion Protein-Based Paper Microfluidics

Since we had to determine baseline conditions of fusion protein-based paper microfluidic devices as a new realm in and of itself, all of our experimentation is foundational and we had to invent the assays to test the devices we created. This trial and error was necessary when exploring protein constructs, paper substrates, channel configurations, and printing methods. After that, we had to test whether our system was viable at all given the conditions we established for it.

*Protein Constructs*

Our fusion protein constructs revolved around three functional domains: a double cellulose binding domain, a super folder GFP, and a hydrophobic domain. By connecting these domains in various configurations using flexible linkers, we synthesized three novel fusion proteins, as well as a past biobrick from Imperial iGEM 2014, that are able to complement each other in different ways. We expand on the design behind each of the novel parts in the biobrick descriptions linked below.
![](https://static.igem.org/mediawiki/2019/9/91/T--BrownStanfordPrinctn--Construct_outline.png)

We were able to synthesize, induce, and extract lysate containing all four of the constructs listed above, and verify their functionality:

\*The functionality of BBa\_K3260031 is showcased in the “Implementing Protein-Based Paper Microfluidics” section below\*

![](https://static.igem.org/mediawiki/2019/9/91/T--BrownStanfordPrinctn--sfGFPLysate.png)

The cell lysate is fluorescing very intensely under UV light, which matches the excitation wavelength of superfolderGFP. This, along with sequencing of the constructs, indicates that we successfully expressed BBa\_K1321348 in lysate. We experimented with all of our constructs suspended in their cell lysate, because it saved time and also all of our constructs contained strong affinity domains that we believed out-competed the natural lysate proteins. In BBa\_K1321348, BBa\_K3260019, and BBa\_K3260021, it is the double cellulose binding domain, and in BBa\_K3260022, it is one half of a strongly complementary leucine zipper pair.

![](https://static.igem.org/mediawiki/2019/f/f9/T--BrownStanfordPrinctn--3634_fluoresence_testing.png)![](https://static.igem.org/mediawiki/2019/a/a6/T--BrownStanfordPrinctn--3634_fluoresence_swatches.png)

Above is a test of the functionality of two of our new fusion proteins, BBa\_K3260036, and BBa\_K3260034. If these two parts assemble correctly on the cellulose surface, there should be fluorescence, which is what we observed here. This means that the leucine zipper components of each protein aligned and held together, allowing the two halves of GFP to stay annealed to the cellulose after washing, thus allowing them complete their chromophore and fluoresce. Because we see fluorescence (highlighted in the color swatch of each sample), the success of the leucine zippers and therefore the functionality of the last two functional domains can be inferred as well, the double cellulose binding domain anchoring the whole complex to the cellulose surface, as well as the hydrophobic domain sticking up from the N-terminal region of BBa\_K3260034, and therefore the assembled protein complex. The successful assembly of these two biobricks not only shows the applicability of these constructs for paper-based microfluidics, but it also shows the potential of split-GFP reporting for fusion proteins in functional applications.

![](https://static.igem.org/mediawiki/2019/8/82/T--BrownStanfordPrinctn--3634_assembly_diagram.png
)

*Paper Substrates*

We tested three main types of “paper" for substrate efficacy: Whatman’s No.4 Filter Paper, Bacterial Cellulose (K. rhaeticus), and archival printer paper. Originally, it was believed that Whatman’s No. 4 chromatography paper would be applicable to the cellulose binding proteins [4]. However, due to some impurities inherent to plant cellulose filter paper (such as the presence of hemicellulose and lignin), and some unpromising results from our preliminary hydrophobicity tests, we decided to try bacterial cellulose from K. rhaeticus. We were able to obtain samples of K. rhaeticus bacterial cellulose from the SoundBio iGEM team in Seattle, Washington (this collaboration is expanded on in our collaborations page). We dried the product out on paper, and on it printed our channel designs (more details on printing below). When we tested our printed proteins on bacterial cellulose, there was no visible capillary action - it seems as if the bacterial cellulose already had some inherent hydrophobic properties that were obscuring the functionality of our hydrophobic proteins.

![](https://static.igem.org/mediawiki/2019/2/2a/T--BrownStanfordPrinctn--whatmansNo4test.png)![](https://static.igem.org/mediawiki/2019/5/55/T--BrownStanfordPrinctn--bacterialcellulose.png)

The top image is of preliminary hydrophobicity tests on Whatman’s No. 4 Filter Paper. The squares were rocked in hydrophobic lysate and dried for varying lengths of time, but when dropping ~2 ul of water on each sample, no increased hydrophobicity or resistance to water was observed between the experimental lysate and control. We then turned to bacterial cellulose of K. rhaeticus which, as mentioned above and displayed in the bottom image, displayed some baseline hydrophobicity that rendered it inapplicable for the functionalization of hydrophobic proteins.

We then decided to try printing our designs on archival grade printer paper. As will be expanded on below, printing our devices on archival paper yielded successful results and was deemed the most promising way forward for developing protein based paper microfluidics.

*Printing Methods*

After trying screen printing and manual painting of proteins, we settled on ‘hacking’ the ink cartridges of commercial printers to print out our protein lysate instead of ink. Our team worked with the Canon Pixma MP950 and the Canon Pixma TR4520 to print our proteins. While the MP950 is a higher end Canon printer, for this particular purpose it was not well suited. Error messages that halted function (that are apparently systemic to all units) were inhibitory when we were trying to make the printer “forget” about tampered parts of its hardware. Also the ink cartridges had reader chips that most likely contained information about the color in the cartridge, and whether it had been used before. For a project that is looking to tamper with cartridges, this was not an ideal characteristic.
The TR4520, on the other hand, worked extremely well for our needs. It held only two cartridges, color (containing compartments with blue, red, and yellow) ink and black ink, which made it extremely easy to control exactly what was being printed. We left the color cartridge intact, and in our print surrounded where we wanted our protein to be (signified with black ink on our printing template) with a bold blue background. This eased visualization of where our protein should have been printed, and also provided a helpful negative control when our testing liquid ran past its final destination in our microfluidic device. This printer was also extremely economical and easy to attain.
We formulated the following general procedure to load and print protein lysate into ink cartridges:

Prepping Cartridge for Lysate Loading:

(used black ink cartridge for protein lysate)

1. Locate the lid, and the seam that connects it to the body of the cartridge.
2. Using a large screwdriver, press into this seam until the cartridge pops off, or can be torn off. Try to make this as clean a separation as possible, as the lid needs to be 3. reattached for adequate loading into the printer.
3. Remove the white sponge from the cartridge, and clean with DIwater until runoff is clear when sponge is squeezed.
4. Rinse inside of cartridge and printhead with DIwater. The printhead is the interface between the sponge and the paper to be printed on. Rinse until runoff from all directions is clear.
5. Let both components dry/ dry off cartridge with paper towel.

Loading Experimental Lysate:

1. Pipette ~1ml of experimental lysate onto printhead pad embedded on the inside of the cartridge. This will aid flow of protein through printhead.
2. Pipette ~2ml of experimental lysate onto depressed square shape on white sponge, making sure it’s well saturated.
3. Pop sponge back into the cartridge, making sure the depressed square on the sponge itself is aligned with the printhead pad.
4. Put lid back on cartridge, using scotch tape to affix it to the body.
5. Load cartridge back into the printer.
6. Go forth and print.

*Designing a Template*

The channel designs we chose to implement to test the viability of microfluidics were based off of Liu et. al [1] where there were “sample zones” in which their liquid would be placed, and then “testing zones” where the liquid would hopefully end up after traversing microfluidic channels of various widths. Their use of a “flower”-based mask allowed us to test a range of channel widths at the same time. We also deconstructed this “flower” to separate the variables we were testing and examine them side by side. The print-out template is shown below, with the dimensions illustrated to the side.

![](https://static.igem.org/mediawiki/2019/d/d0/T--BrownStanfordPrinctn--template_diagram_measurements.png)

The dimensions we chose were meant to show the range of channels normally encountered in both wax channel microfluidics and wider range PDMS devices. We found these designs to cover the full range of our protein functionality.
Since we commandeered the black ink cartridge for our experimental lysate, we outlined our desired microfluidic channels in black ink in out file to print out. Then, to delineate where the protein was being printed and where it wasn’t, we surrounded our protein “zone” with blue, which would be printed out as normal printer ink.

*Implementing Protein-Based Paper Microfluidics*

As mentioned above, the implementation of archival printing paper into our printer system yielded positive results for our protein-based microfluidic devices. To test our microfluidic devices, we used water with green food-coloring.

BBa\_K3260031 Single Layer Print:

[
 ](https://static.igem.org/mediawiki/2019/2/22/T--BrownStanfordPrinctn--test_one.mov)

The video above shows one of our tests with printing with protein lysate. The lysate that we loaded into the black ink cartridge contained BBa\_K3260031(our dCBD + our hydrophobic, or Radek) domain. Directly below is a labelling of this video with channel widths for each component in our testing template.

![](https://static.igem.org/mediawiki/2019/a/a6/T--BrownStanfordPrinctn--video_one_labels.png)

The blue regions are plain printer ink, while to the white sections in between the blue and the black outline of the channels are printed protein lysate. We can see on the range of straight, separated channels that the water efficiently wicked up the 250um wide channel and the 200um channel. On the largest “flower” template, the water flowed up the 800um and 900um channels. We also see a successful serendipitous negative control of our device once the water flows past the end of one of the 900um channels and starts penetrating the paper printed with blue ink. This provides a nice contrast with the channel flow and illustrates that while the resolution of the channels isn’t extremely sharp, the hydrophobic protein is certainly performing its function. On the smaller scale flower channels, the water efficiently travels down the 100 - 500 um channels, which is indicative of its functionality on the PDMS-scale resolution.

BBa\_K3260031 Double Layer and Negative Control Side by Side:

[
 ](https://static.igem.org/mediawiki/2019/9/9f/T--BrownStanfordPrinctn--test_two.mov)

The video above is another printing lysate test. This test print was part negative control (single layer, but underprinted with normal ink) and part double layer printed BBa\_K3260031, which is a dCBD fused to a single hydrophobic domain. Below is a labelling of the video with channel width for each component in our testing template.

![](https://static.igem.org/mediawiki/2019/f/fa/T--BrownStanfordPrinctn--video_two_labels.png)

On this test we have a firmer grasp on the resolution that is achieved with this printing method, and it is shown that some bleeding into the hydrophobic region of the device design occurs. However, this is sharply contrasted with the complete lack of resolution and function in the negative control, that was single layer printed with our hydrophobic protein but printed on the opposite side with plain blue ink, which undercuts the functionality of the protein. We can see that the water effectively travels up the channels of widths 200 um, 250 um, 800 um, and 900 um.

*Conclusion*

All in all, we were able to rationally design, synthesize, print, and prototype a microfluidic system whose channel architecture is based around fusion proteins and paper reagents, not wax or PDMS. We were able to create functional microfluidic channels down to 100um in width, which is extremely significant because this falls comfortably within the range of high resolution PDMS device channels (which have widths of 30 nm to 500 um [2, 3]). Even though our channels did experience a significant degree of bleeding, the functionality of their wicking ability even down to 100um widths speaks to their success as a proof of concept. In addition to this, we were able to produce these prototypes using low cost commercial printers, ink cartridges, and paper reagents. Not only did we successfully produce these prototypes with our original fusion proteins idea (BBa\_K3260031), but we also experimented with more elaborate protein designs, yielding a fusion system that can be applicable to our paper microfluidic system, but also an extremely wide range of applications involving the functionalization of cellulose with a binding domain, split GFP domains, and complementary leucine zippers (illustrated in BBa\_K3260036 and K3260034). Coming back to the central goal of the subproject, this proof of concept for a protein-based paper microfluidic system opens the door for a whole new range of low cost, easily producible, high resolution devices.

1. Liu, Ning, et al. “Direct Spraying Method for Fabrication of Paper-Based Microfluidic Devices.” Semantic Scholar , Nanyang Technological University , Sept. 2017.
2. Abidin, Ummikalsom, et al. “Replication and Leakage Test of Polydimethylsiloxane (PDMS) Microfluidics Channel.” AIP Publishing, AIP Publishing LLC, 25 Jan. 2019, aip.scitation.org/doi/abs/10.1063/1.5086611.
3. Strong, E. Brandon, et al. “Fabrication of Miniaturized Paper-Based Microfluidic Devices (MicroPADs).” Nature News, Nature Publishing Group, 9 Jan. 2019, www.nature.com/articles/s41598-018-37029-0.
4. Sher , Mazhar, et al. “Paper-Based Analytical Devices for Clinical Diagnosis: Recent Advances in the Fabrication Techniques and Sensing Mechanisms.” HHS Public Access, Molecular Diagnostics , 26 July 2017.

### Production: In-Lab Drug Production

For the cell-free expression system, the pET-23b(+) plasmid was successfully linearized (Figure 1) and the overhangs for Gibson assembly were successfully added for the hG-CSF and teriparatide inserts (Figure 2). The Gibson assembly of the hG-CSF insert into the linearized plasmid was verified by sequence analysis. For the *B. subtilis* cellular system, pBS2E was successfully linearized (Figure 3) and Gibson assembly was verified to be successfully through sequence analysis. For a control, the chromogenic protein Prancer Purple from ATUM Paintbox was successfully linearized for Gibson into pET-23b(+) (Figure 4).

![](https://static.igem.org/mediawiki/2019/5/58/T--BrownStanfordPrinctn--pet23b_lin.png)

Figure 1. Linearization of pET-23b(-) (expected size ~3524bp).

![](https://static.igem.org/mediawiki/2019/6/6e/T--BrownStanfordPrinctn--drug_lin.png)

Figure 2. hG-CSF with 6x his tag and overhangs (expected size ~1257bp) and teriparatide with 6x his tag and overhangs (expected size ~360bp)

![](https://static.igem.org/mediawiki/2019/a/a6/T--BrownStanfordPrinctn--pBS2E_lin.png)

Figure 3. pBS2E plasmid linearized for Gibson transformation (expected size 6262bp).

![](https://static.igem.org/mediawiki/2019/3/38/T--BrownStanfordPrinctn--prancer_purple_lin.png)

Figure 4. Prancer Purple (ATUM Paintbox) cut out from plasmid (expected size 687bp).

After we got our sequencing results and confirmed that we had constructs for pET23-teriparatide-EK, pET23-teriparatide-TEV, pET16-teriparatide-TEV, and pET16-hGCSF-TEV, we moved on to expressing and purifying them in *E. coli*  and cell-free reactions. *E. coli*  expression and subsequent His-tag purification gave inconclusive results due to large amounts of native protein making the drug bands unclear. The cell-free process is discussed below.

### Establishing a Cell-free System

We made all the mixtures required for the cell-free reactions, and extracted lysate from *E. coli* BL21 (DE3) cells using the protocols here. To test for contamination, we streaked 50uL aliquots of each solution onto LB plates and incubated them at 37° °C overnight. The amino acid mix, salt mix, energy mix, did not have any contamination; an abundance of growth from the lysate aliquot indicated contamination, which was undesirable (Fig. 1). To remove the unlysed cells from our solution, we modified the protocol by adding an extra centrifugation step in smaller volumes, and at higher speeds. The result of our modification lead to decreased in-tact bacteria presence from an uncountable amount of colonies per 50uL to 62 ± 5 colonies per 50uL Figure 2.

![](https://static.igem.org/mediawiki/2019/5/5e/T--BrownStanfordPrinctn--cfr_Figure_1.jpg)

![](https://static.igem.org/mediawiki/2019/8/8a/T--BrownStanfordPrinctn--cfr_Figure_2.jpg)

With our mixtures and solutions in place, we ran preliminary cell-free experiment using an Red Fluorescent Protein-encoding plasmid to test if our platform worked at all. After leaving the cell-free reaction to incubate overnight, we qualitatively observed production of RFP (Fig. 3). At this point, we did not collect quantitative fluorescence values. The red arrows pointing at experimental groups that produced a small amount of fluorescence and the blue arrows pointing to the negative control (no plasmid inserted). In Figure 4 we see our positive control, where we expressed the RFP in *E. coli*. The cellular-expression positive control is significantly brighter than the cell-free experimental group. This may be because of the greater concentration of RFP present in the positive control. From this data, we knew that RFP production was observable.

![](https://static.igem.org/mediawiki/2019/archive/c/c5/20191022003633%21T--BrownStanfordPrinctn--cfr_Figure_3.jpg)

![](https://static.igem.org/mediawiki/2019/1/15/T--BrownStanfordPrinctn--cfr_Figure_4.jpg)

Given the initial encouraging results we set to establish our first set of cell-free experiments to see if we could enhance the reaction by manipulating concentrations of T7 Polymerase and Murine Inhibitor on cell-free, and see if anything significantly helps protein yield, or if anything is not as essential, so we can avoid lyophilizing it. The reactions were run for 5hrs. Afterwards, the fluorescence of the samples was measured using the spectrophotometer. Figure 5 shows qualitative results of the experiment.

![](https://static.igem.org/mediawiki/2019/5/54/T--BrownStanfordPrinctn--cfr_Figure_5.jpg)

There is a clear indication that the experiments did not work. The fluorescent data for the experimental groups was well below the controls (data not shown), indicating there was no fluorescence, hence no protein product at all. Under UV light, there was also no observed red color that was evident in the initial experiment.

We began our lengthy troubleshooting process. There were many points to begin from: an ineffective lysate batch, DNA plasmid quality and concentration, environmental contamination by RNases, low protein activity, decomposition/precipitation of cell-free reaction mixes, degradation of the solutions at room temperature while preparing the reactions.

First we tested if changes in T7 Polymerase quantities would influence the reaction, so we doubled the concentration from 10U to 20U, but observed no RFP production, and because we observed no fluorescence under UV light, we did not attempt to quantify our results using the spectrophotometer.

Then we suspected there might be problems with the DNA, so we began by altering the DNA concentration by testing adding 100ng of plasmid to the reaction instead of the previous 30ng, and testing one of the paper-microfluidic protein constructs that contained a green fluorescent protein (GFP). Both experiments did not yield any observable red or green fluorescence.

New cell lysates were extracted and cell-free mixes were remade in case of any chemical degradation or contamination that may have occurred to render the solutions dysfunctional, and reactions were run again. Again, there was no obvious production of RFP.

Another problem with the DNA was contamination. We only received a very small quantity of RFP plasmid from iGEM, so we had miniprepped it. Miniprep DNA tends to contain a lot of nucleases, and these nucleases, RNases in particular, inhibit cell-free reactions. To diminish RNase activity, we employed Murine RNase inhibitor, and tried to keep our lab environment sterile and RNase free. The experiments we ran that manipulated RNase quantity also yielded no production of RFP. However, it is possible we underestimated the degree of contamination and did not add enough RNase inhibitor. Our SDS-PAGE highlights the samples that contained RNase Murine inhibitor in blue versus the samples that did not contain RNase inhibitor in red. There is no observed production of any protein besides those latent in the solution. At this point, we had also received a commercial cell-free kit, and began to test reactions on it. One of the commercial cell-free reactions we ran is R1 (Fig. 6) - no production of RFP was observed either despite the edition of Murine RNase inhibitor.

![](https://static.igem.org/mediawiki/2019/e/e9/T--BrownStanfordPrinctn--cfr_Figure_6.jpg)

Realizing that we were underestimating the amount of Murine RNase inhibitor we needed for the reaction, we increased the quantity in a commercial cell-free trial from 20U of Murine to 100U of Murine, and observed a band in the 27-30kDa range, which corresponded to the size of RFP (Fig. 7). This is also reflected in the positive control sample (expression of RFP in *E. coli*).

![](https://static.igem.org/mediawiki/2019/a/ab/T--BrownStanfordPrinctn--cfr_Figure_7.jpg)

While this result was promising, we could not replicate it consistently, so we had to continue reiterating through the trouble-shooting process. We tested different control plasmids and our peptide-drug plasmids too. During one reaction, we were able to successfully produce teriparatide in a commercial cell-free system (Fig. 8).

![](https://static.igem.org/mediawiki/2019/c/c0/T--BrownStanfordPrinctn--cfr_Figure_8.jpg)

In tandem, we also tried to establish a yeast cell-free system that could perform the post translational modifications required for the insulin. However, we came across the same problems as the *E. coli* system, and weren’t able to observe anything qualitatively or quantitatively.

Despite promising initial results with our lab-characterized cell-free expression system, we were unable to get it to preform consistently most likely due to excess RNase contamination, and degradation of unstable compounds in the mixes. Likewise, we were unable to get the commercial cell-free system to preform consistently for the same reasons. If our experimentation process had gone more smoothly, we would have scaled down our cell-free experiments to fit inside the microfluidic expression chip, and manipulated concentrations of T7 Polymerase, RNase inhibitor, DNA concentration, and incubation time to enhance peptide-drug production to our desired concentrations.

### Expression and Purification Microfluidics

For the drug purification system, a full set of chips to produce and purify medicine were designed, fabricated and tested. This included a 3D printed cell free expression chip, a column for Immobilized Metal Affinity Chromatography (IMAC), and a column for Size Exclusion Chromatography (SEC). All of our chips were fully fluid functional.

![](https://static.igem.org/mediawiki/2019/0/0c/T--BrownStanfordPrinctn--ExpressionChipPrinted.jpg)

Finished expression chip interfaced with tubing and a syringe

In order to tackle existing constraints on the minimum feature capabilities of our 3D printer, we used several different approaches to manufacture our expression chip, falling into two main catagories. The first group of iterations was a design that required us to print two halves of our expression chip, and use photo-activated resin to seal them together. After these failed due to leaking and misalignment, we iterated again and printed the chip as a single piece. With some minor adjustments to our design and protocol we were successful: its interfaces were water-tight and we were able to successfully facilitate protein-expression by flowing through the expression chip.
![](https://static.igem.org/mediawiki/2019/2/2f/T--BrownStanfordPrinctn--IMACPrototype.jpg)

Finished IMAC column interfaced with syringe pump

![](https://static.igem.org/mediawiki/2019/3/38/T--BrownStanfordPrinctn--SECPrototype.jpg)

Finished SEC column interfaced with syringe pump

These chips were then flow tested to see if there were any significant issues with bubbling, leakage or delamination of the substrate. We found we had manufactured several fluid functional chips for each of our 3 designs. Videos of our chips being flow tested are shown below.

[

](https://static.igem.org/mediawiki/2019/9/99/T--BrownStanfordPrinctn--IMACFlowTest.mov)

Flow test for IMAC column using blue food coloring to show liquid more clearly

[

](https://static.igem.org/mediawiki/2019/2/21/T--BrownStanfordPrinctn--ExpressionFlowTest.mov)

Flow test for expression chip replacing blue food coloring with red food coloring

While some difficulties were encountered loading chromatography resin in our initial prototypes, we were able to confirm the concept behind the purification modules we created by running a large scale size exclusion chromatography to separate yellow food coloring from an orange chromoprotein.

![](https://static.igem.org/mediawiki/2019/c/c6/T--BrownStanfordPrinctn--SECFractions.jpeg)

Fractions collected from large scale SEC. Initial 2ml fractions are clearer, then become more yellow over time, qualitatively demonstrating that small yellow food coloring molecules were not present in initial fractions.

![](https://static.igem.org/mediawiki/2019/a/ab/T--BrownStanfordPrinctn--SECFractionsUV.jpeg)

SEC fractions shown under UV light. Initial fractions that were clear under normal circumstances fluoresced under UV while the last 2 fractions did not, indicating that the larger chromoprotein eluted in the first two fractions. This qualitatively demonstrates how our SEC column could be used to separate proteins from small molecules

After testing SEC purification on a large scale, we returned to our microfluidic designs and created a new prototype with an enlarged media loading port and multiple layers of smaller bead blockers. With this new design we were able to load SEC media into our prototype.

[

](https://static.igem.org/mediawiki/2019/3/3d/T--BrownStanfordPrinctn--SECLoading.mov)

SEC column with media being loaded

The main difficulties encountered with our second set of prototypes were that the IMAC channel height of 100um was too low for the beads in the media, which caused them to not be able to enter the chip. While the SEC could be loaded, its input and output channels were small enough that some beads that passed through our bead blockers could still clog channels. Future prototypes for this will need to have both taller channels and wider inlet and outlet channels

Our results with microfluidics showed very promising results. Overall we were able to mill and test 2 fluid functional PDMS microfluidics, and 1 3D printed chip. All of our chips were fully fluid functional, and some significant strides were made in adding chromatography media to our chips. Our hardware reached a similar level of development to many other excellent iGEM projects which focused on microfluidics. BostonU 2017 created a repository of microfluidic devices, which were milled, tested and fluid functional, but not tested on biologics. Our team has fluid tested our chips to the same extent, although we also encountered difficulties incorporating biologics on our chips. UCopenhagen 2018 also sought to create a drug purification system that would work in space, and was able to 3D print several versions of their hardware. We were also able to 3D print hardware for drug production, although both of our teams encountered difficulties incorporating hardware into a fully realized drug production and purification system. Overall our team made significant strides in developing microfluidic hardware that could someday represent a major breakthrough in the way drugs are produced, both in space and on Earth.

### Lyophilization

We preformed three lyophilization experiments. In the first experiment, we freeze-dried *B. subtilis* in growth media on plastic, *E. coli* in growth media on plastic surface, *E. coli* in growth media on a filter paper surface, and *E. coli* in growth media on printer paper. After overnight lyophilization, we rehydrated the cells at different time points (t=0d, 1d, 2d, 8d, 10d, 15d, 30d) and preformed serial dilutions to calculate cell viability. Some of the data is omitted due to contamination of growth plates and procedural errors.

Our results show that *B. subtilis* has consistent growth even at the one month time point. This is expected because *B. subtilis* are hardy spore-forming bacteria known to have survive for up to six years in space [1]. However, *E. coli* viability dramatically decreases one day after lyophilization on paper and plastic substrates when no lycoprotectant is present (fig. 1.). Hence, we turned began testing lycoprotectants to see if them can increase cell viability when freeze-dried.

![](https://static.igem.org/mediawiki/2019/d/dd/T--BrownStanfordPrinctn--lyophilizedvmax.png)

Next, we tested different concentrations of trehalose as a lycoprotectant for *E. coli*. There were four experimental groups: 0% Trehalose, 1% Trehalose, 5% Trehalose, and 10% Trehalose. The samples were lyophilized overnight and rehydrated at time points t = 0d, 1d, 7d, 15d, after which serial dilution experiments were preformed to calculate cell viability.
We observed that *E. coli* in the 0% Trehalose condition exhibited the best cell viability overtime, which is contrary to the previous results and to the literature. However, on rehydration at t = 0d, the 10% Trehlose condition had the greatest CFU count compared to all the other groups (fig. 2), which suggest that Trehalose had the best inital viability, but procedural or lyophilzation errors may have results in its quick deterioration.

![](https://static.igem.org/mediawiki/2019/d/d9/T--BrownStanfordPrinctn--lyophilizedtrehalose.png)

In our third lyophilization experiment, we tested the preservation ability of skim milk, sucrose, saline, and growth media on VmaxTM. Not much growth was observed at the two rehydration time points (t = 0d,1d), except for a few colonies under the sucrose condition at t = 0d (Fig. 3). These results were also unexpected, but were attributed to errors in the experimental procedures; during this time, we were encountering problems with VmaxTM growth from the manufacturers end, so it is possible that the cells themselves were not viable in the first place.

![](https://static.igem.org/mediawiki/2019/7/79/T--BrownStanfordPrinctn--lyophilizedvmaxreal.png)

It is interesting that the cell viability for many of the experimental groups decreases a few days after lyophilization. While this dramatic decrease could be attributed to keeping our freeze-dried samples at room temperature, which encourages degradation, it is possible that there were errors in our freeze-drying procedure that inhibited true exploration of lyophilization capabilities. We left our experiments lyophilize overnight (around 10-14hrs), whereas other procedures lyophilize for 24 to 48hrs; there is the possibility that not all the water was removed from our samples. Additionally, not all freeze-drying experiments simply let the samples sit in the lyophilizer – there are more intricate cycles of freezing and drying, and seperationg of unstable compounds that we were unable to conduct due to laboratory and time constraints [4].

While we did attempt to freeze-dry cell-free solutions, none of the results are reliable because the solutions prior to lyophilization may not have been functional given the difficulties we had in trying to develop our cell-free system. If our cell-free system was operating smoothly, we would

Overall, our lyophilization confirm that *B. subtilis* would be the most storable chassis for cellular expression of peptide drugs for the Astropharmacy because their cells were consistently viable over an extended period of time at room temperature. However, we were unsuccessful in replicating long-term viability in *E. Coli* and VmaxTM despite our use of lycoprotectants suggested by literature. This may be because our experiments were rather sporadic and not as systematic as they could have been. The experiments should have focused on a single species surveying various conditions to establish a more defined protocol that could then be applied and modified to different species. Future experiments would have investigated other modes of drying (like desiccation), and further tested lyophilization on paper to enhance the manufacturing and transportability of a lyophilized Astropharmacy on a paper-based system.

1. *Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate*. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct\_ooreos. Accessed 20 Oct. 2019.
2. ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
3. Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
4. Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
5. Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
6. Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
7. Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
8. Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
9. Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
10. Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
11. Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
12. Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.