iGEM 2019 — Team Penn

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# Team:Penn/Notebook

[Home](https://2019.igem.org/Team%3APenn)

Team

[Team Members](https://2019.igem.org/Team%3APenn/Team)
[Attributions](https://2019.igem.org/Team%3APenn/Attributions)

Project

[Inspiration](https://2019.igem.org/Team%3APenn/Description)
[Wax Printing](https://2019.igem.org/Team%3APenn/WaxPrinting)
[Design](https://2019.igem.org/Team%3APenn/Design)
[Hardware](https://2019.igem.org/Team%3APenn/Hardware)
[Assembly](https://2019.igem.org/Team%3APenn/Assembly)
[Notebook](https://2019.igem.org/Team%3APenn/Notebook)

Results

[Characterization](https://2019.igem.org/Team%3APenn/Results)
[Demonstrate](https://2019.igem.org/Team%3APenn/Demonstrate)

Public Engagement

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

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

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

![](https://static.igem.org/mediawiki/2019/7/7c/T--Penn--logo_square_no_bg.png)

# MIYAGI:Notebook

# Open Source Wax Printing Conversion Kit

Week 1: June 3 - June 9

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We spent the week researching different methods of fabricating microfluidic paper analytical devices (uPADs). We weighed the pros and cons of different fabrication methods such as photolithography and wax screen printing. We looked into the differences between piezoelectric and thermal printheads. We also considered various possible substrates and hydrophobic substances for making low cost uPADs.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware We took apart a Xerox ColorQube 8580 wax printer to understand how the printer deposits the wax. This printer has been used to design uPADs and is used in the bioengineering lab course at the University of Pennsylvania. While it allows rapid prototyping and is easy to use, the printer and its cartridges are expensive and is no longer manufactured. This type of uPAD fabrication also allows limited resolution because once the wax is printed onto the paper, the paper must be heated to allow the wax to fully permeate through the paper. This heating process causes the wax to spread into the channels limiting the resolution.

Week 2: June 10 - June 16

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We reached out to a Philadelphia local startup, Chip Diagnostics, to get insights and feedback on our design for our wax printer.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Replicating open source projects

We began experimenting with different XY table designs. One design incorporated a pulley system and stepper motors to create a vertical plotter for drawing on a
whiteboard. Another design was taken from an open source XY table used for depositing liquid into a microwell plate. We adapted it to fit our needs to create a general XY table.

![](https://static.igem.org/mediawiki/2019/0/0e/T--Penn--xyplotter.png)

Vertical XY plotter

![](https://static.igem.org/mediawiki/2019/7/77/T--Penn--xytable1.png)

Open source XY table

![](https://static.igem.org/mediawiki/2019/0/05/T--Penn--xytable2.jpeg)

Our XY table

Taking apart existing projects

We took apart a crayola crayon melter and a wax jewelry gun to analyze the heating elements and extrusion mechanisms. The crayon melter uses a heating element and a temperature feedback system that rises to ~160 F to heat regular crayola crayons that are made of paraffin wax. The extrusion mechanism was a large ball point that had to be pressed down to the paper to extrude the molten wax in thick streams.

![](https://static.igem.org/mediawiki/2019/c/ce/T--Penn--crayonmelter2.jpeg)

Inside of the Crayola Crayon melter

![](https://static.igem.org/mediawiki/2019/7/79/T--Penn--crayonmelter11.png)

Using the Crayon Melter

![](https://static.igem.org/mediawiki/2019/3/3d/T--Penn--jewlery1.jpeg)

Inside a wax jewelry gun

Week 3: June 17 - June 23

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We met with Evan Weinstein, CEO and founder of local 3D chocolate printing startup Cocoa Press, to discuss the difficulties associated with building a CNC machine from scratch and to gain insight into the design process. Evan gave us recommendations on which hardware components are most commonly used and which firmware programs have the best documentation.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Replicating open source projects

The XY table project was completed. We were successfully able to control the movement of a marker in 2 dimensions by using G-code to control stepper motors. Through this project we learned the hardware and software fundamentals of CNC machining.
Heating pads were used to heat a syringe to the point of melting the wax inside. As recommended by the representative from Chip Diagnostics, we used adhesive motorcycle hand warmers to achieve even contact around the cylindrical syringe body.
We assembled a Prusa i3 MK3S to be used in the University of Pennsylvania George H. Stephenson Foundation Undergraduate Laboratory and Bio-Makerspace. This open source 3D printer is considered the industry standard for desktop 3D printing and costs $750 in parts.

![](https://static.igem.org/mediawiki/2019/5/5c/T--Penn--completedXY.png)

Completed XY Table

![](https://static.igem.org/mediawiki/2019/e/ed/T--Penn--syringe1.jpeg)

Heating a Syringe with Motorcycle Warmers

![](https://static.igem.org/mediawiki/2019/9/90/T--Penn--Prusa1.png)

The Prusa i3 MkS3

PIVOT

After considering the cost of building an XY table compared to the position accuracy we would likely be able to achieve, we decided to modify an existing open source 3D printer into a wax printer instead of designing a custom XY stage. In recent years, 3D printer costs have steadily decreased while printer quality has increased. Many research facilities and universities now have access to 3D printers. Our new plan was to make a kit to convert the Prusa i3 MK3S into a wax printer capable of making paper based microfluidic devices. Research groups with access to a Prusa printer would then only need to source parts for the conversion kit, saving time and money compared to building or buying a new wax printer. Research groups without access to a similar 3D printer would need to purchase more parts, but at a price similar to the expected cost for our original design.

Week 4: June 24 - June 30

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research Most extrusion based printers are either powered by a mechanical pump or a pneumatic system. After weighing the pros and cons of each mechanism, we decided that the pneumatic system was better for our purpose. We anticipate that using a pneumatic powered syringe pump will allow more precise control of the wax extrusion.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Heating

We started experimenting with motorcycle heating pads and nichrome wire as our heating elements. We have wrapped the motorcycle heating pads around 50 ml syringes and used thermistors and feedback control to regulate the syringe temperature. We have also tried wrapping nichrome wire around the syringes, however the nichrome wire becomes too hot and melts the plastic. Without any insulation or covering over the syringe, it takes a long time for the syringe to get hot and actually melt wax. The wax we are using is Sigma-Aldrich Paraffin wax with a melting temperature of 53-58 C.

Week 5: July 1 - July 7

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We met with Jordan Miller, a previous University of Pennsylvania professor and iGEM advisor, to speak about his work with the BariCUDA Extruder, an open source air pressure driven extruder. Dr. Miller uses his design for precise pneumatic extrusion of sugar filaments for plating living vascular tissues. We spoke with him about our design considerations and received input about in regards to heating our device and using a pressure system to drive the extrusion mechanism.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Syringe Holder

Uniform heating is a crucial element for extruding molten wax and preventing clogging of the syringe. To ensure the syringe is heated we made a CAD design of a metal syringe holder that our syringe will be placed inside of. The metal syringe holder will be wrapped in cylindrical heating pads so as to conduct heat more efficiently to the syringe full of wax. We met with the director of Penn’s machine shop to discuss design considerations and decided on aluminum for our design as it was a low cost metal that provided the heat conductance we wanted.

![](https://static.igem.org/mediawiki/2019/4/46/T--Penn--cadfile1.png)

Pneumatic System

We started experimenting with a 3 way 2 position solenoid valve that we plan to use for the pneumatic system. The solenoid valve switches between pressurized air and atmospheric air. We are using a nitrogen tank to provide the pressurized air. Tubing will connect the nitrogen tank to the solenoid valve. We 3D printed a syringe adapter that will allow the tubing to connect the solenoid valve to the syringe.When the solenoid is powered on, it connects the pressurized air to the syringe resulting in extrusion. When the solenoid valve is powered off, it connects atmospheric air to the syringe resulting in no extrusion. To extrude molten wax, only 5-10 psi is needed. To incorporate the solenoid valve into our device, we have experimented using the fan port of the Prusa. Since our design does not necessitate a fan, we will use the fan port to turn the solenoid valve on and off.

Week 6: July 8 - July 14

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We looked into how the Prusa regulates the temperature of the plastic it is extruding. The Prusa relies on PID (proportional integral derivative) control which can be easily edited using the Prusa firmware. We plan on using the Prusa’s internal PID tuning and setting our own desired temperature.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Software

Before you can print an object on a 3D printer you first need an stl file that is then converted into G-code. G-code serves as a set of instructions for the machine and tells the printhead where to move, how fast to move, what path to follow, etc. A slicer program is used to convert stl files to G-code, the Prusa printers use the PrusaSlicer program. We have edited a python script from the Jordan Miller lab that converts the G-code from the PrusaSlicer into G-code that is needed to print our wax uPADs. The program edits the G-code so that all extrusion commands are replaced with commands to turn on the solenoid valve. We tested the conversion script and upload the edited G-code to the Prusa. We replaced the solenoid valve with an LED so that every time the solenoid was turned on, the LED would turn on as a visual indicator that the script was working.

Week 7: July 15 - July 21

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Syringe Holder

We updated the syringe holder design to include a full cylinder design instead of a tapered head at the syringe tip. Having a full cylinder will lower costs of the machined part as well as make it easier to adhere the heating pads to the outside of the syringe holder.

Pneumatic System

We experimented with extruding molten wax with the pressure system. However, melted paraffin wax has such a low viscosity, the wax just drips out of the tip when no pressure is being applied. We plan on trying smaller gauge syringe needles and potentially may implement a negative pressure system to prevent dripping.

Week 8: July 22 - July 28

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Pneumatic System

We tested smaller gauge syringe tips with the pneumatic extrusion system. We tried 20, 22, and 25 gauge syringe tips. The 25 gauge syringe tip worked the best and had the least amount of wax dripping out however became clogged the most frequently. Only about 1 drop of wax leaked out every ~ 10 seconds. We ordered smaller gauge needles and are also thinking of transitioning to shorter needles to reduce the amount of time the wax spends in the needle and prevent clogging.

Week 9: July 29 - August 4

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Heating

We experimented with new heating pads. Instead of using the motorcycle heating pads we started using heating pads from adafruit that are smaller and easier to wire to the Prusa. The replaced the 24V extruder heating prt on the Prusa Arduino mega with two 12V heating pads in series. We edited the firmware of the Prusa to override the usual heating settings and input our own. We successfully PID tuned the Prusa to heat to 65 C.

Week 10: August 5 - August 11

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Heating

We printed our first uPADs. Our main issues are that too much wax is extruded onto the paper, the syringe tip gets clogged, and the extruder head is too far from the print bed. To decreases the amount of wax being extruded we plan on trying smaller syringe tips, reducing the pressure, changing the syringe volume, increasing the feed rate, and experimenting with more viscous waxes.

Syringe Holder

We sent updated syringe holder designs to the machine shop for smaller volume syringes and smaller gauge syringe tips.

Week 11: August 12 - August 18

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We tested the effects of the melting point of wax on its extrusion to determine the best type of wax for our device. We found that the melting point of wax does not have any large effects on the extrusion of the wax from the syringe.

Week 12: August 19 - August 25

![](https://static.igem.org/mediawiki/2019/d/d7/T--Penn--hardware_icon.png)

#### Hardware

Hardware

We designed and printed a final syringe mount that fully supports the extruder. We installed this mount onto the Prusa printer with screws and fit the syringe and holder onto it.

Pneumatic System

We ordered and received a smaller pressure gauge to regulate our input pressure to smaller values. We then tested the extrusion at smaller pressures.

Week 13-16: August 26 - September 22

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research With all of the finalized parts, we performed experiments to determine the specifications of the printer. We printed many variations with wax onto cellulose filter papers and performed post-hoc measurements to calculate the resolution and reliability of the printer.

Week 17-20: September 23 - October 20

![]( https://static.igem.org/mediawiki/2019/e/ee/T--Penn--research_icon.png)

#### Research We optimized the G-code to produce successful prints taking into consideration the dripping and pooling of wax. We then printed paper microfluidic devices of different designs onto the cellulose filter papers.


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# Team:Penn/Experiments

[Home](https://2019.igem.org/Team%3APenn)

Team

[Team Members](https://2019.igem.org/Team%3APenn/Team)
[Attributions](https://2019.igem.org/Team%3APenn/Attributions)

Project

[Inspiration](https://2019.igem.org/Team%3APenn/Description)
[Wax Printing](https://2019.igem.org/Team%3APenn/WaxPrinting)
[Design](https://2019.igem.org/Team%3APenn/Design)
[Hardware](https://2019.igem.org/Team%3APenn/Hardware)
[Assembly](https://2019.igem.org/Team%3APenn/Assembly)
[Notebook](https://2019.igem.org/Team%3APenn/Notebook)

Results

[Characterization](https://2019.igem.org/Team%3APenn/Results)
[Demonstrate](https://2019.igem.org/Team%3APenn/Demonstrate)

Public Engagement

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

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

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

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Penn/Results

[Home](https://2019.igem.org/Team%3APenn)

Team

[Team Members](https://2019.igem.org/Team%3APenn/Team)
[Attributions](https://2019.igem.org/Team%3APenn/Attributions)

Project

[Inspiration](https://2019.igem.org/Team%3APenn/Description)
[Wax Printing](https://2019.igem.org/Team%3APenn/WaxPrinting)
[Design](https://2019.igem.org/Team%3APenn/Design)
[Hardware](https://2019.igem.org/Team%3APenn/Hardware)
[Assembly](https://2019.igem.org/Team%3APenn/Assembly)
[Notebook](https://2019.igem.org/Team%3APenn/Notebook)

Results

[Characterization](https://2019.igem.org/Team%3APenn/Results)
[Demonstrate](https://2019.igem.org/Team%3APenn/Demonstrate)

Public Engagement

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

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

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

# **Results**

Characterizing our device

# Characterizing Wax Droplets

###### Since droplets are the minimum outputs of our wax printer, we first investigated the property of wax drops to characterize the performance of our printer and optimize mechanical and electrical settings for better performance.

###### Extrusion of wax droplets was conducted by applying pressure for a set amount of time in the scale of milliseconds. To examine dependent variables of droplet properties, we altered three physical parameters: the extruder temperature, the amount of pressure, and the duration of pressure. We printed 96 drops for each setting and quantified them by measuring their sizes and weights, as well as calculating the success rates of extrusion. At the same time, we briefly assessed how well wax droplets permeate through the filter paper.

###### **Fig1** shows how the size and weight of wax droplets change with altered duration of pressure, where the temperature and the amount of pressure were fixed to 80 [℃] and 5.0 [psi] respectively. It is demonstrated that the volume of wax droplets increases as the duration of pressure increases. A similar trend was observed when the amount of pressure was varied while the duration and temperature was fixed.

![](https://static.igem.org/mediawiki/2019/5/5f/T--Penn--waxdrops_size.png)

###### While printing wax droplets, we found that droplets sometimes do not get extruded or get extruded with extra wax from a previous extrusion, so we calculated the rates of wax droplets to be successfully extruded. We defined the extrusion to fail when there is no extrusion occurring or when the extruded droplet is twice as big as other droplets. The success rate is highly correlated to the pressure pulse length but is not highly correlated to the amount of pressure. This is probably because all of the pressure we tested was above the minimum amount needed to extrude wax from our device. Also, little or no correlation was seen between the success rate and the temperature when testing temperatures higher than 80 [℃].

![](https://static.igem.org/mediawiki/2019/c/c0/T--Penn--waxdrops_successrate_color1.png)

**Fig2.** Surface plot of successful extrusion rate (0.0-1.0) where duration or amount of pressure was changed between 20[ms] and 50[ms] or 5.0 [psi] and 10.0 [psi] respectively. Temperature was set to 80 [℃].

###### Next, we tested how wax permeates through the paper (Whatman®︎ qualitative filter paper, grade 595) at different extrusion temperatures. **Fig.3** shows pictures taken from the backside of filter papers where wax droplets (light blue) were printed and dyed water (dark blue) flowed through. As shown in those pictures, wax permeates better when the temperature is higher presumably due to the increased fluidity. Although increasing temperature resulted in larger droplets on paper, it also reduced the minimum extrudable pressure from 4.0 [psi] (80℃) to 2.5 [psi] (90℃), which compensates for the resolution loss (**Fig3C**).

![](https://static.igem.org/mediawiki/2019/9/9f/T--Penn--wax_5psi_80c.png)
![](https://static.igem.org/mediawiki/2019/a/a9/T--Penn--wax_5psi_90c.png)
![](https://static.igem.org/mediawiki/2019/6/6e/T--Penn--wax_3psi_90c.png)

**Fig3.** Pictures taken from the backside of paper where wax droplets were printed and dyed water flowed afterwards. Areas where droplets did not permeate through the paper are shown with white circles. A) temperature: 80 [℃] pressure: 5.0 [psi], B) temperature: 90 [℃] pressure: 5.0 [psi], C) temperature: 90 [℃] pressure: 3.0 [psi].

# Characterizing Wax Lines

###### Extruding wax lines with high consistency and resolution is crucial to print functional paper microfluidic devices. We compared two different extruding methods to draw lines: continuous extrusion and drop-wise extrusion. Using each method, our printer either continuously extrudes the stream of wax or intermittently emits wax droplets onto the paper while moving, as if printing pixels.

###### We first optimized parameters for drop-wise extrusion. In addition to the parameters investigated during characterization, we altered another parameter, the interval of drops. Pictures were taken of drop-wise printed wax lines and, consequently, those pictures were converted into binary images. The linewidth was measured by using our custom ImageJ plugin function that counts pixels at 500 different points of each line, and its median was determined. The line completeness was defined as the probability of the counted pixels not being 0 within 500 values.

###### Increasing the interval of droplets merely helps printing thinner lines because of the huge variation in the line width itself (**Fig4** Left). Another problem is the trade-off between line resolution and continuation of drops: when the interval of drops is longer, the resolution is higher, but the line itself tends to be more discontinuous (**Fig4** Right). Theoretically, there should be an optimal interval for this trade-off, but the success rate of wax drop (~95%) was seemingly too low to find the sweet spot.

![](https://static.igem.org/mediawiki/2019/3/3f/T--Penn--dropwise_line.png)
![](https://static.igem.org/mediawiki/2019/b/b4/T--Penn--dropwise_discontinuity.png)

**Fig4.** The amount of pressure, duration, or temperature was set to 3.0[psi], 30 [ms], or 90 [℃] respectively. Left) Each data point represents the median linewidth within each line. The line and the band represent linear regression and its 95% confidence intervals. Right) Each data point represents the line completion within each line. The line and the band represent logistic regression and its 95% confidence intervals.

###### For continuous extrusion we took an additional parameter, the speed of the extruder movement, into consideration. With increased printing speed, line width logarithmically decreased with reasonably small variations among printing processes(**Fig. 5**).

###### To note, one drawback of using the continuous extrusion method is that hot liquid wax could move as the print bed moves and spread around the print area. This happens because the printing speed for the continuous method is sometimes too fast for the wax to get solidified before the next print bed movement. The problem could be solved by modifying our munging script to optimize the printing process.

![](https://static.igem.org/mediawiki/2019/9/90/T--Penn--continuous_line.png)

**Fig5.** Each data point represents the median linewidth within each line. The line and the band represent the logarithmic fitting curve and its 95% confidence intervals. Throughout the experiment, the amount of pressure was fixed to 3.0 [psi], and temperature was set to 90 [℃].

###### In order to comprehensively compare different extrusion methods and parameters, we analyzed printed lines to determine three features: resolution, consistency, and completeness. The resolution, consistency, and completeness were estimated by measuring the median linewidth, the coefficient of variation of the median linewidth, and the line completion, respectively. Table 1 summarizes those features for potential sets of extrusion method and parameters. Between the two extrusion methods, the continuous method is better in many aspects and among different parameters, the printing speed of 10000 [mm/min] was chosen as best for having high consistency, line completeness, and resolution compared to others.

![](https://static.igem.org/mediawiki/2019/2/24/T--Penn--line_summary.png)

**Table 1.** Summary of statistical features of lines printed under different conditions. Lines to measure statistical features were independently printed for three times (N=3). The average linewidth was defined as mean ± standard deviation of three median linewidth. The coefficient of variation was determined by dividing the variance of three median linewidth by the mean value.

# Characterizing Channels

###### Using the continuous extrusion method with optimized parameters, we further investigated how closely two lines could align to create a hydrophilic channel, which is a key component of microfluidics. Printed channels were analyzed by following our line width analysis method, except that dyed water was applied to each channel so as to make binary images.

###### Although channel completion requires at least 5mm of designed channel width to reliably create microfluidic channels, designed width and actual width of channels clearly demonstrate a linear relationship. For these investigations, we concluded that the resolution limit of our current version of wax printer is 5 [mm], and our resolution loss from the ideal line is about 2 [mm].

![](https://static.igem.org/mediawiki/2019/8/8a/T--Penn--channel_width.png)
![](https://static.igem.org/mediawiki/2019/b/ba/T--Penn--channel.png)

**Fig6.** Left) Each data point represents the median channel width within each printed channel. The solid line and band show the linear regression line and its 95% confidence intervals. The dashed line shows ideal correspondence (y=x) between the designed width and printed width. Right) Each data point represents the channel completeness (0.0-1.0) for each printed channel. The fitted line and band show the logistic regression line and its 95% confidence intervals.

# Printing Functional Devices

###### **Fig7**shows a microfluidic device printed with our assembled wax printer. In order to avoid using low-quality sections of wax lines, we further modified our munging script. It inserts resting commands when the extruder rapidly changes its direction of movement.

###### First, our munging code virtually sets the circle depicted as a dashed line in the figures, whose points later become resting areas for the extruder when it pauses extruding. The code inserts resting commands whenever angles between lines exceeds the implemented threshold (>60[deg] in **Fig7**), while avoiding lines in the design area.

![](https://static.igem.org/mediawiki/2019/8/88/T--Penn--demo_drawing.gif)
![](https://static.igem.org/mediawiki/2019/b/ba/T--Penn--demo.png)
![](https://static.igem.org/mediawiki/2019/c/c2/T--Penn--demo_actual.png)

**Fig7.** Top Left) Virtual demonstration of wax printing. Solid lines represent where wax is extruded, solid points represent where the extruder pauses, and the circle drawn with dashed lines shows where the potential resting areas are. Top Right) Figure of device using the code depicted within the animation. Bottom) Picture of printed device.

# Future Directions

###### **Dripping Problem:** Throughout the project, a major issue has been melted wax dripping from the syringe tip when not extruding. In future iterations, negative pressure in the syringe could be used to prevent unwanted dripping. Although we did not present results, we found that the relative height of the extruder correlates with the quality of extrusion. Therefore, if the dripping problem could be solved, we would be able to test lower extruder height and potentially improve the overall quality of extrusion.

###### **Syringe Tip Size:** Besides dripping, another major issue has been the clogging of syringe tips, especially at smaller diameters. Assuming that clogging was caused by the narrow path and lower temperature at the edge of the tip, the problem could be potentially solved by further improving heat conductivity.

###### **Dragging Wax:** There is another wax printing method we have not tested. By dragging wax onto the paper while extruding, the printer can potentially draw fine lines as the extruder works basically like a fountain pen. When we accidentally dragged lines when setting the extruder height very low, we observed lines with quite high resolution and consistency.

# Penn iGEM 2019

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