This assignment was the first time for both Xinyin and I to work with 3D printing. It was a fresh and exciting experience, and through multiple attempts, we learned a lot.

After discussion, we decided to make the Cas9 molecule from Streptococcus pyogenes (SpCas9) for the following reasons: First, this molecule is what Xinyin likes and is currently working with. Second, Xinyin had some experience in using PDB and Pymol. Although it seems challenging to make, we thought it should be achievable. Lastly, if it is successfully created, this will be a good tool for future teaching.

Our first step was downloading the protein structure PDB file from PDB (https://www.rcsb.org/structure/4CMP). Then, we imported the PDB file into Pymol and converted it into a molecule structure. By watching this video (https://www.youtube.com/watch?v=VNU6YHFi7Lg), we successfully exported the molecule image into the STL file format. The picture below was the molecular pattern we tried to print for the first time:

We chose the Bambu Machine (FDM printing) to print our molecule for two reasons: its cost was cheaper than SLA and other methods, and its support structure was easier to remove. However, we were not sure what size was appropriate. We then tried two different length settings, three and four inches, and chose the tree support. The picture below were our settings before printing:

Initially, the printing process was not smooth, and we encountered some error messages. Fortunately, there were TAs or lab assistants on site who could help us solve the problems. The picture below shows some error messages we saw:

We stayed at the printer until the first few layers were successfully printed. The next day, when we went to pick up the finished products, we were delighted to see that our molecules had been successfully printed. After we removed the support materials on both products, we decided that the 4-inch one was our best size choice because many support materials in the three-inch product were challenging to remove completely.

 

We decided to print another 4-inch molecule since we must make two identical models for this assignment. This time, we kept the same settings but changed the support type from tree to normal support to see if the normal support would be more straightforward to remove.

Unfortunately, the normal support was more difficult to remove because so many tiny fibers were stuck in the small holes of our molecule. We finally gave up removing all the support materials. On the same day, we discovered that our original protein structure model contained two chains of Cas9. For scientific accuracy and having some previous printing experience, we decided to redo our work again, making two new Cas9 molecules by separating the two chains and printing them together in the same file. By watching the same video as mentioned above (https://www.youtube.com/watch?v=VNU6YHFi7Lg), we were able to separate two chains and print them again. This incident taught us that we should be more careful in confirming the model before starting printing so as not to waste more time later. The video and pictures below are our new separated structure and printing settings:

These two molecules took 7 hours and 46 minutes to print. Then, our post-processing work began. It took the two of us half an hour to remove all the support material. Because of the characteristics of our molecules, we did not plan to sand them but directly color them. We applied the Vallejo paint as accurately as possible according to different domain ranges, which took us about 2 hours to finish. After painting, our last step was to spray the clear spray paint to give our products a glossy appearance.

The picture below is our final product:

Cost table for the TWO 3D-printed spCas9 

Cost Type	Cost	Price	Source	Quantity	Total
Materials	PLA filament	$19.99 / kg	Bambu Lab	0.0845 kg	$1.69
	Vallejo Paints	$4.13 / Fl Oz	Amazon.com	0.12 Fl Oz*	$0.50
	Spray paint-clear	$0.5 / Oz	Homedepot	0.1 Oz	$0.05
Labor	3D printing Lab Assistant	$17.5 / hour**	Glassdoor.com	0.25 hr***	$4.38
	Prototyping Engineer (Intern)	$17 / hour	ZipRecruiter.com	6.5 hr	$113.90
Overhead	Bambu 3D printer (rental price)	$19/ day	Fatllama.com	8 hr	$6.33
	Electricity (for the light, the computer, and the printer)	$0.13 per kWh	Raise3D	2.3 kWh	$0.3
	Post-processing tools (tweezers and brushes)	Tweezer $5.29 each Brushes $3.99/ set	Amazon.com Amazon.com	1 set	$9.28
	Quality control	27.5/ hour******	LinkedIn.com	0.5 hr	$13.75
Design	Engineering and Development*****	$17 / hour	ZipRecruiter.com	1 hr	$17.00
	Iterations *****	$17 / hour	ZipRecruiter.com	1 hr	$17.00
Misc.	Waste and Scrap	$0.66/Gal= $0.17/L	Houstontx.gov	0.025L	$0.0042

Sum: $184.1842

*We used 70 drops of Vallejo Paints, and we estimate each drop to contain 0.0016907 Fl Oz.

**We took the average of $14 and $21.

***The lab assistant replaced the filament, addressed error messages, and took out the products. The hours they spend on the products are at most 15 minutes.

****0.5 hours for setting up the printing and monitoring. For each of two people, 0.5 hours for removing the support, and 2.5 hours for coloring and spraying, 0.1 hours for cleaning. 0.5+2x(0.5+2.5+0.1) = 6.7 hours

*****We treat it as a part of the intern time.

******We took the average of $25 and $30.

Here is a picture of our clean table after work: