Individual Final Project: SALEM II: Synthetic Animated Luminescent Engineered Microbes

(Note: Basing this format on https://2026a.htgaa.org/2026a/course-pages/final-projects/individual/index.html )

SECTION 1: ABSTRACT:

Large-format Animated Opentrons Art

The idea here is to create large format and animated Opentrons art mosaics, by developing my lab automation and fluorescent bacteria skills to the point where I can create larger format artwork from multiple petri dishes. Shown above is a fairly quick test I made of trying to design rotating DNA strands in seven petri dishes of Opentrons agar art. For this, my art/design will emphasize the colors that show up best in fluorescent bacteria art; for example, green, red, and blue. My main synthetic biology challenges are 1) Can I create new custom color(s) biodesigned by me, based on existing mScarlet? and 2) Can I design fluorescent proteins with hidden DNA messages from my ancestors?

I am descended from women executed during the Salem Witch Trials, so I carry part of their DNA with me. I am calling this project “SALEM: Synthetic Animated Luminescent Engineered Microbes.” (My original workimg title was “Something Something Sacred Sigil System.”)

I aim to engineer fluorescent protein variant(s) based on my ancestor Mary Eastey who was executed at the Salem Witch trials. These will be mutants based on mScarlet, will include a quote from Mary Eastey encoded into their DNA, may be spectrally shifted toward or into invisible-to-humans infrared, and may be fused with NanoLuciferase for bioluminescence. (This may represent three initial designs for me to test.)

The quote I will embed into the DNA is from Mary Eastey’s final petition to the court, “I petition to your honours not for my own life for I know I must die and my appointed time is set … if it be possible no more Innocent blood may be shed.” From the University of Virginia’s Salem Witch Trials Documentary Archive https://salem.lib.virginia.edu/n45.html#n45.22

I will create custom designs for fluorescent bacteria colors and Opentrons python code using the “Fluorescent Pixel Art” tools created by HTGAA TA Ronan Donavan. See https://opentrons-art.rcdonovan.com/

As shown below, the design of the petri dish setup – six circles around a single center circle – is basically what is described as the “Egg of Life” of sacred geometry. The original chart on the left below is from https://pardesco.com/blogs/news/sacred-geometry-art-symbols-meanings

The animation system where a still image becoem anoimated when it is rotated is based on the “phenakistoscope,” an early pre-film motion picture device created in the 1830s. The phenakistoscope is sort of like a flip book, but instead of flipping through pages to creates the illusion of motion, you spin a disc with images. You can see more info at https://publicdomainreview.org/collection/phenakistoscopes-1833/

And below is a historical drawing from the History of Science Museum in the UK https://www.mhs.ox.ac.uk/exhibits/fancy-names-and-fun-toys/phenakistiscopes/index.html, showing how a phenakistoscope was viewed, through slots on the disc with the image reflected in a mirrror.

The phenakistoscope is a precursor to the more well known “zoetrope,” where the motion picture frames are arranged on the inside of a cylinder rather than on a disc. “Zoetrope” is based on the Greek words for “wheel of life.”

So, the basic idea here is to create a sacred geometry “egg of life,” that animates like a zoetropic “wheel of life,” based on DNA, the “blueprint of life,” containing convicted “witch” Mary Eastey’s quote “I petition to your honours not for my own life …”

Significance … Broad Objective … Hypothesis … Specific Aims … Methods …

SECTION 2: PROJECT AIMS:

Aim 1: Experimental Aim:

Engineer and test fluorescent protein variant(s) based on my ancestor Mary Eastey who was executed at the Salem Witch trials. My initial “aim one” is to will be to create DNA containing a quote from Mary Eastey inside a generic construct, which will also contains a red flurescent protein based on mScarlet.

I will design these synthetic biological experimental colors in Asimov Kernel and Benchling, and I will order from them from Twist Bioscience. I will initially test the color(s) by hand by streaking on agar with inoculating loop in the BUGSS Lab.

Future designs may include mutant colors spectrally shifted toward or into invisible-to-humans infrared, and may be fused with NanoLuciferase for bioluminescence.

Aim 2: Development Aim:

Possible further development aims include:

• Create the ultimate design itself
• Create the Opentrons liquid handling roboto code
• Design and create devices for live rotating display, with correct speed and lighting, rather than digital rotation
• Design possible systems for time lapse animation, during the printing proces and/or growing process.
• Maybe phosphorescent bacteria that eat other phosphorescent bacteria over time …
• Longer form animation, with multiple sets of petri dishes that form longer video(s).

Thanks to HTGAA BUGSS crew Amanda, Joel, Juhi, Mantis, Violeta, and Marian with whom I discussed these aims (some of these are their ideas!)

Aim 3: Visionary Aim:

Maybe: Live performance of Opentrons art. Below should be an embeded video clip from a previous performance with live-coded video and sound art, with my friend, mentor and collaborator Wes Taylor, professor at Wayne State University. A next-level aim for this project might be live-coded performances with large-format animated Opentrons art.

SECTION 3: BACKGROUND:

• Briefly summarize two peer-reviewed research citations relevant to your research (minimum four sentences).
• Explain how your project is novel or innovative. (Minimum 3 sentences.)
• Explain why your project matters and what impact it could have. (Minimum 5 sentences.)
• Describe the ethical implications associated with your project and identify relevant ethical principles (e.g., non-maleficence, beneficence, justice, or responsibility). (Minimum 2 paragraphs.)

SECTION 4: EXPERIMENTAL DESIGN, TECHNIQUES, TOOLS, AND TECHNOLOGY

Create a detailed experimental plan for your final project. Include a timeline for each part of your experimental plan (i.e., how long you would expect each step in your final project to take).

1. I need to convert the quote from Mary Eastey into a DNA sequence.

One way would be to first convert the sentence into letters available as IUPAC amino acid codes – https://www.bioinformatics.org/sms/iupac.html – which only has 20 letters, so I changed every B to a P, every O to a Q, every U to a V, and removed spaces:

IPETITIQNTQYQVRHQNQVRSNQTFQRMYQWNLIFEFQRIKNQWIMVSTDIEANDMYAPPQINTEDTIMEISSETIFITPEPQSSIPLENQMQREINNQCENTPLQQDMAYPESHED

Then using a reverse translate – https://www.bioinformatics.org/sms2/rev_trans.html – I converted that to a sequence of most likely codons:

attccggaaaccattaccattcagaacacccagtatcaggtgcgccatcagaaccaggtg cgcagcaaccagacctttcagcgcatgtatcagtggaacctgatttttgaatttcagcgc attaaaaaccagtggattatggtgagcaccgatattgaagcgaacgatatgtatgcgccg ccgcagattaacaccgaagataccattatggaaattagcagcgaaaccatttttattacc ccggaaccgcagagcagcattccgctggaaaaccagatgcagcgcgaaattaacaaccag tgcgaaaacaccccgctgcagcaggatatggcgtatccggaaagccatgaagat

I then double checked that with with a Translate tool https://www.bioinformatics.org/sms2/translate.html to confirm it translates back into:

IPETITIQNTQYQVRHQNQVRSNQTFQRMYQWNLIFEFQRIKNQWIMVSTDIEANDMYAP PQINTEDTIMEISSETIFITPEPQSSIPLENQMQREINNQCENTPLQQDMAYPESHED

Note that there are number of other ways to encode messages in DNA – https://earthsciweb.org/js/bio/dna-writer/ – https://www.cachesleuth.com/dnacode.html – https://dnacode.bc.cas.cz/index.php?ln=en – but I think I prefer spelling with the IUPAC amino acid codes rather than an aribitrary code system. This way, any synthetic biologist who can read the protein sequence can decode it.

2. I need to get the fluorescent protein to combine this with

I am thinking mScarlet-I3-NCwt – https://www.fpbase.org/protein/1VSM7/ – since it is 1) Scarlet and there are many occult scarlet references, 2) I3 is almost the number 13, which is the typical number of witches in a coven, etc.; and 3) NCwt is almost “newt” as in “eye of newt” from the witches’ potion chant of Shakespeare’s “MacBeth. “In the caldron boil and bake; Eye of newt and toe of frog, Wool of bat and tongue of dog … For a charm of powerful trouble, Like a hell-broth boil and bubble.”

The sequence of mScarlet-I3-NCwt is:

MVSKGEAVIK EFMRFKVHME GSMNGHEFEI EGEGEGRPYE GTQTAKLKVT KGGPLPFSWD ILSPQFMYGS RAFIKHPADI PDYWKQSFPE GFKWERVMIF EDGGTVSVTQ DTSLEDGTLI YKVKLRGGNF PPDGPVMQKR TMGWEASTER LYPEDVVLKG DIKMALRLKD GGRYLADFKT TYKAKKPVQM PGAFNIDRKL DITSHNEDYT VVEQYERSVA RHSTGGMDEL YK

3. I need to design this, preferably in Asimov Kernel …

Here was my construct design in Asimov Kernel as of April 15, 2026, this is now out of date! I am keeping it here for documention and explanation purposes:

From left to right, this early version had:

• T7 promoter
• A1 RBS
• Millikin Eastey Test 1 (my code insert)
• BBa_K4654001 (mScarlet-I3 red fluorescent reporter; I can switch this to mScarlet-I3-NCwt)
• T7 Terminator
• BBa_K4235018 (Ampicillin Resistance Gene)
• BBa_K4411019 (pET28a-backbone)

I checked this out with our BUGSS lab crew and asked if everything seemed good. Our awesome TA Amanda said maybe Kanamycin resistance was what we used before. So, I was thinking about switching Ampicillin Resistance Gene for Kanamycin Resistance Gene, and then I looked up the pET28a-backbone and according the IGEM Registry of Standard Biological Parts at https://parts.igem.org/Part:BBa_K3521004 “pET28a-Backbone … contains anti-kanamycin genes.” So, I am likely getting rid of any resistance genes in my construct since they seem redundant with what the pET28a-backbone already has.

Updated construct plan April 17, 2026:

From left to right, this most recent version has:

• T7 promoter
• A1 RBS
• Millikin Eastey Test 2 Attribution (my code insert, now with MARYEASTEYERICMILLIKIN attribution after the quote, which you can see at the bottom of the screenshot above)
• BBa_K3183014 (Gly-Ser-Gly Protein Domain Linker for E. coli)
• mScarlet-I3-NCwt (red fluorescent protein from https://www.fpbase.org/protein/1VSM7/)
• T7 Terminator
• BBa_K4411019 (pET28a-backbone)

I ran some simulations in Asimov Kernel. The screenshots below show RNAP Flux and Ribosome Flux seemingly pretty well-balanced, and the protein concentration over time seems fast enough that experiment results are pretty quick, but not so fast that there would be worries that something toxic might grow too quickly.

I ran Asimov Kernels’ Biosecurity sequence scanner using SecureDNA. Below is a screenshot showing “No flagged sequences detected.”

And I exported from Asimov Kernel using “Construct as GenBank” and that file should be available at Millikin-SALEM-Construct-Eastey-Quote.gb

UPDATE construct plan April 22, 2026:

Met with awesome Lisa Scheifele at BUGSS Lab Executive Director who helped me improve this! Basically, I still had soem redundancies between my Asimov Kernel construct and the pET28a-backbone/

This is my updated Mary Eastey code, CDS (-start -stop) for Asimov Kernel:

    1 attccggaaa ccattaccat tcagaatacc cagtatcagg ttcgtcatca gaatcaggtt
   61 cgtagcaatc agacctttca gcgtatgtat cagtggaatc tgatttttga atttcagcgt
  121 attaaaaatc agtggattat ggttagcacc gatattgaag ctaatgatat gtatgctccg
  181 ccgcagatta ataccgaaga taccattatg gaaattagca gcgaaaccat ttttattacc
  241 ccggaaccgc agagcagcat tccgctggaa aatcagatgc agcgtgaaat taataatcag
  301 tgcgaaaata ccccgctgca gcaggatatg gcttatccgg aaagccatga agatatggct
  361 cgttatgaag ctagcaccga atatgaacgt atttgcatga ttctgctgat taaaattaat

And this is my mScarletI3NCwt CDS (-start +stop) for Asimov Kernel:

    1 atggttagca aaggtgaagc tgttattaaa gaatttatgc gttttaaagt tcatatggaa
   61 ggtagcatga atggtcatga atttgaaatt gaaggtgaag gtgaaggtcg tccgtatgaa
  121 ggtacccaga ccgctaaact gaaagttacc aaaggtggtc cgctgccgtt tagctgggat
  181 attctgagcc cgcagtttat gtatggtagc cgtgctttta ttaaacatcc ggctgatatt
  241 ccggattatt ggaaacagag ctttccggaa ggttttaaat gggaacgtgt tatgattttt
  301 gaagatggtg gtaccgttag cgttacccag gataccagcc tggaagatgg taccctgatt
  361 tataaagtta aactgcgtgg tggtaatttt ccgccggatg gtccggttat gcagaaacgt
  421 accatgggtt gggaagctag caccgaacgt ctgtatccgg aagatgttgt tctgaaaggt
  481 gatattaaaa tggctctgcg tctgaaagat ggtggtcgtt atctggctga ttttaaaacc
  541 acctataaag ctaaaaaacc ggttcagatg ccgggtgctt ttaatattga tcgtaaactg
  601 gatattacca gccataatga agattatacc gttgttgaac agtatgaacg tagcgttgct
  661 cgtcatagca ccggtggtat ggatgaactg tataaataa

And this is my construct design from Asimov Kernel, much simpler than before, as before I was including paets that were redendant with the PET-28a backbone.

While making my constructs, I referenced Asimov Kernel’s bacterial demos at https://kernel.asimov.com/htgaa-2026/repositories/repository/692aad68-86f9-47e5-9655-d35dc4122b26 as well as examples of other fluorescent protein plasmids like https://www.addgene.org/189757/

4. Place Twist Bioscience order.

I put it into the spreadsheet ON DATE at https://docs.google.com/spreadsheets/d/1UsoGDb2TVIM-r8aScY800lMelQP_JPp1pCr5CB4y0zM/edit?gid=882133822#gid=882133822 and also notified my node leads need to submit a form that is linked at the top of that Google spreadsheet.

6. Test the color(s) by hand by streaking on agar with inoculating loop
7. Finalize a design for 7 petri dishes

I had been planning on using the Fluorescent Pixel Art tool at https://opentrons-art.rcdonovan.com/

It has been updated for Ginkgo cloud lab use, so I am developing my own system to convert images into color coordinates … writing soem scripts to run in terminal using Image Magick …

My design for the center petri dish has a star made of arrows, based on the SBOL visual glyph for promoter https://sbolstandard.org/docs/SBOL-Visual-2.2.pdf as well as the chaos star of Michael Moorcock fantasy novels and the later used in chaos magick by Peter J. Carroll.

8. Use Opentrons liquid handling robot to create the artwork
9. Rotate in digital video editing to test the animation effect

Some of the synthetic biology techniques most relevant to my project

• Bioethical Considerations
• DNA Gel Art: DNA Editing
• DNA Gel Art: DNA Construct Design
• DNA Gel Art: Databases
• Lab Automation: Creating Code for Laboratory Automation
• Lab Automation: Using Liquid Handling Robots (e.g., Opentrons)
• Lab Automation: Designing a Twist Order
• Protein Design: Use of Asimov Kernel
• Protein Design: Use of Benchling
• Protein Design: Databases

Expand upon two techniques you checked in the previous question by describing how you would utilize those techniques in your final project. (min. 4 sentences)

Identify any How To Grow (Almost) Anything Industry Council companies which are associated with your final project (optional)

Associated with Aim 1 of my final project would probably be:

• Asimov Kernel https://www.asimov.com/kernel is likely where I design much of my new fluorescent proteins
• Opentrons https://opentrons.com/ is what I will use for automated pipetting of the designs
• Twist Bioscience https://www.twistbioscience.com/ is where i will order the DNA

Associated with possible Aim 2 and Aim 3 would be those related to biomaterials like maybe:

• Biofabricate https://www.biofabricate.co/ “sustainable biomaterial innovation for all sectors”
• BIOME Consortia https://www.biomeconsortia.org/ “cutting-edge technology to unlock and apply the near-infinite potential of microorganisms”
• Bolt https://boltthreads.com/ “B-silkTM is inspired by a special silk produced by spiders”
• Epibone https://www.epibone.com/ “facial bone product … customized for patients via a CT scan … with stem cells extracted from patients’ abdominal fat”
• Mycoworks https://www.mycoworks.com/ “create a platform for the highest quality materials using Fine Mycelium”
• Upside Foods https://upsidefoods.com/ “Cultivated meat is meat grown directly from animal cells”

SECTION 5: Results & Quantitative Expectations

SECTION 6: ADDITIONAL INFORMATION

List all references cited in this assignment (bullet-point list)

• Predatory Bacteria Are Fierce, Ballistic and Full of Potential - https://www.scientificamerican.com/article/predatory-bacteria-are-fierce-ballistic-and-full-of-potential/
• Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo - https://pubs.acs.org/doi/10.1021/jacs.2c02320
• The Xenotext Experiment: An Interview with Christian Bök - https://pmc.iath.virginia.edu/issue.107/17.2voyce.html
• Infinite Transformations in a Suitcase: Encountering Human-DNA Interaction through Poetry-infused Wine -https://dl.acm.org/doi/10.1145/3689050.3707674
• Eduardo Kac’s “Cypher, 2009 - https://www.ekac.org/cypher.text-info.html

Create a supply list and budget for your project (bullet-point list)

• BUGSS Lab membership (cover lab sace and supplies)
• Twist order (my custom DNA constructs)

Group Final Project