Week 2 HW: DNA Read, Write, and Edit

Part 1

Benchling & In-silico Gel Art

Part 2

No wet lab access

Part 3

DNA Design Challenge

Choose Protein

I chose the amino acid sequence of VioC - Chromobacterium violaceum for Violacein pigment.

I will reverse translate and codon optimize to amplify pigment production and thus its antimicrobial, UV-resistant properties.

sp|Q9S3U9|VIOC_CHRVO Violacein synthase OS=Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / CCUG 213 / NBRC 12614 / NCIMB 9131 / NCTC 9757 / MK) OX=243365 GN=vioC PE=1 SV=2 MKRAIIVGGGLAGGLTAIYLAKRGYEVHVVEKRGDPLRDLSSYVDVVSSRAIGVSMTVRG IKSVLAAGIPRAELDACGEPIVAMAFSVGGQYRMRELKPLEDFRPLSLNRAAFQKLLNKY ANLAGVRYYFEHKCLDVDLDGKSVLIQGKDGQPQRLQGDMIIGADGAHSAVRQAMQSGLR RFEFQQTFFRHGYKTLVLPDAQALGYRKDTLYFFGMDSGGLFAGRAATIPDGSVSIAVCL PYSGSPSLTTTDEPTMRAFFDRYFGGLPRDARDEMLRQFLAKPSNDLINVRSSTFHYKGN VLLLGDAAHATAPFLGQGMNMALEDARTFVELLDRHQGDQDKAFPEFTELRKVQADAMQD MARANYDVLSCSNPIFFMRARYTRYMHSKFPGLYPPDMAEKLYFTSEPYDRLQQIQRKQN VWYKIGRVN

Reverse translate

sp|Q9S3U9|VIOC_CHRVO Violacein synthase OS=Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / CCUG 213 / NBRC 12614 / NCIMB 9131 / NCTC 9757 / MK) OX=243365 GN=vioC PE=1 SV=2 ATGAAGCGAGCGATTATTGTCGGGGGGGGTTTAGCTGGAGGTCTAACTGCGATATACTTGGCTAAACGTGGATACGAGGT ACATGTGGTCGAGAAACGGGGCGACCCACTCAGGGACCTGTCTAGCTATGTTGATGTGGTTTCATCACGCGCAATCGGGG TCAGCATGACTGTAAGAGGCATCAAGTCAGTTTTAGCGGCCGGTATCCCCCGAGCTGAATTAGATGCCTGTGGTGAGCCA ATAGTTGCCATGGCGTTTTCCGTCGGGGGACAATATCGCATGCGGGAACTTAAACCACTCGAAGACTTCCGACCGCTTTC GCTTAACCGAGCAGCCTTCCAGAAGCTTTTGAACAAGTACGCAAACCTTGCCGGCGTACGGTACTATTTCGAACATAAAT GCCTGGATGTAGACCTGGATGGGAAATCCGTACTGATCCAAGGGAAGGACGGACAGCCGCAGCGACTTCAAGGAGATATG ATTATCGGCGCAGATGGGGCACACAGTGCAGTTCGCCAAGCGATGCAGTCAGGATTGCGGCGCTTTGAGTTTCAACAAAC GTTCTTTAGGCACGGGTATAAAACGCTGGTCCTACCCGACGCCCAAGCACTCGGGTATCGAAAGGACACGTTATATTTTT TTGGAATGGACAGCGGAGGGTTGTTCGCAGGCCGAGCCGCAACAATACCCGATGGTAGCGTGTCCATAGCTGTGTGTCTG CCCTACTCCGGCTCCCCCAGTTTGACAACCACAGATGAACCGACTATGCGTGCATTTTTCGACAGGTACTTTGGAGGTCT TCCACGGGATGCGAGGGACGAGATGCTTAGACAATTTTTAGCCAAGCCGTCTAATGATCTAATAAATGTGCGATCTTCAA CTTTTCATTACAAAGGTAACGTTCTGCTTTTAGGCGACGCCGCACATGCTACCGCGCCATTTTTAGGACAAGGCATGAAT ATGGCGTTAGAGGATGCGCGAACATTCGTAGAATTACTTGATCGCCACCAAGGCGATCAGGATAAAGCGTTTCCAGAGTT CACGGAGCTTAGAAAGGTGCAAGCGGACGCGATGCAAGATATGGCCCGGGCGAATTACGATGTTCTATCTTGCTCCAACC CGATTTTTTTTATGAGGGCGCGGTATACCCGCTACATGCACAGCAAGTTTCCGGGACTGTACCCGCCGGATATGGCCGAG AAACTGTATTTCACGTCAGAGCCGTACGATCGATTACAACAAATACAGCGCAAGCAAAACGTATGGTACAAGATAGGCAG AGTTAAT

Codon Optimize

https://en.vectorbuilder.com/tool/codon-optimization/b93b7790-7536-4d9b-a72e-02d62c3944e8.html

Next Next steps would be to embed into a seaweed matrix.

Part 4

Prepare a Twist DNA Synthesis Order

After reading more on living materials, bacterial pigments, and connecting it to my interest in light and circadian rhythms, I wanted to explore how to make a simple biological system that expresses anti-microbial or other elements only when needed, rather than all the time. So building a ’temporal’ antimicrobial system that produces a bacteria-killing peptide Magainin on a 24-hour schedule controlled by a circadian promoter RpaA. I started with just learning how to design the Magainin peptide and annotate properly.

Benchling

Twist

REF:

  1. Fang et al. (2025) - “Mechanism and reconstitution of circadian transcription in cyanobacteria”
  2. Salis et al. (2009) - “Automated Design of Synthetic Ribosome Binding Sites”
  3. Westerhoff et al. (2008) - “Structure, Membrane Orientation, Mechanism, and Function of Pexiganan (Magainin derivative)”

Part 5

DNA Read/Write/Edit

DNA Read (Sequencing)

What DNA would you want to sequence and why?

I would sequence my pLight-Circadian-Color plasmid (which contains the RpaA gene from Synechococcus elongatus, an anthocyanin color gene, and a light sensor) to check that it was made correctly before testing if bacteria with this plasmid change color on a 24-hour schedule when exposed to light.

What sequencing technology would you use?

I would use Sanger sequencing because it’s most accurate.

DNA Write (Synthesis)

What DNA would you synthesize and why?

I would synthesize my yet-to-be-completed pLight-Circadian-Color plasmid containing three genes (RpaA from Synechococcus elongatus for timing, anthocyanin for color, light sensor for activation) to test if bacteria can change color on a 24-hour schedule in response to light.

DNA Edit

What DNA would you edit and why?

After I verify the plasmid works, I would edit the RpaA promoter to make it stronger so the color changes are brighter and more noticeable on a 24-hour schedule.

What editing technology would you use?

I would use site-directed mutagenesis to make small changes to the RpaA promoter because it’s precise.

References & Resources

Lecture Materials

  • Week 2 Lecture - DNA Read, Write, & Edit, George Church, Joe Jacobson, Emily Leproust
  • Week 2 Lab - DNA Gel Art, February 12-13, 2026

Required Readings

  1. Fang et al. (2025). “Mechanism and reconstitution of circadian transcription in cyanobacteria.” Journal of Biological Chemistry
  2. Salis et al. (2009). “Automated Design of Synthetic Ribosome Binding Sites to Control Protein Expression.” Nature Biotechnology, 27, 946-950
  3. Westerhoff et al. (2008). “Structure, Membrane Orientation, Mechanism, and Function of Pexiganan (Magainin derivative).” Biochemistry

Software & Tools Used

  • Benchling - DNA sequence design, annotation, and in-silico gel electrophoresis
  • Twist Bioscience - DNA synthesis order preparation and optimization
  • VectorBuilder Codon Optimization Tool - Reverse translation and codon optimization for violacein synthase
  • UniProt - Protein sequence database (VioC entry: sp|Q9S3U9|VIOC_CHRVO)
  • Imgur - Image hosting for documentation

Sequences Worked With

  • VioC (Violacein synthase) from Chromobacterium violaceum strain ATCC 12472
  • RpaA circadian promoter from Synechococcus elongatus
  • Magainin antimicrobial peptide sequence

AI Assistance

  • Claude (Anthropic) - DNA design and sequencing strategy
    • Model: Claude Sonnet 4.5
    • Date(s) used: February, 2026
    • Tasks: Assisted with reverse translation strategy for VioC, guidance on codon optimization principles, clarified Sanger sequencing vs synthesis tradeoffs

Project Development

  • Circadian-controlled antimicrobial system design (RpaA + Magainin)
  • Violacein pigment amplification through codon optimization
  • pLight-Circadian-Color plasmid conceptual design

Additional Resources

  • Twist Bioscience synthesis guidelines and specifications
  • Benchling annotation standards
  • Circadian rhythm gene expression literature

Acknowledgments

  • Course instructors
  • TAs for Benchling platform guidance