Week 2 HW: DNA Design

This design advances my Week 1 proposal for structurally coloured cellulose films. I want a black, melanin-containing bacterial-cellulose backing to deepen the contrast of a separate colour-producing layer. For this assignment, I assembled a Tyr1 tyrosinase expression cassette in Kernel. The digital construct is a proposed component for a future prototype; I have not built or tested a pigmented film (Baig, 2026; Walker et al., 2025).

Kernel workspace

I used the HTGAA 2026 workspace and the baig-growingcolours repository. The saved construct is named J23104-B0034-tyr1-B0015 melanin cassette. Figure 1 shows it in Kernel (Asimov, 2026).

1. Expression target

The target is Tyr1, a 297-amino-acid tyrosinase derived from Bacillus megaterium. Tyr1 catalyses oxidation of L-tyrosine toward melanin formation. I would use the dark pigment as the backing to the cellulose-based colour layer (National Center for Biotechnology Information [NCBI], n.d.; Walker et al., 2025).

The proposed chassis is Komagataeibacter rhaeticus. This bacterial-cellulose producer was part of my Week 1 proposal and has been studied as a host for engineered cellulose and melanin production (Baig, 2026; Florea et al., 2016; Walker et al., 2025).

2. Parts and vector context

The 1,076 bp Kernel construct contains the expression cassette. The selected pSEVA331Bb backbone is a proposed build target and is not part of the saved Kernel cassette.

3. Assembled cassette and reading frame

The CDS has 297 sense codons followed by one stop codon. Kernel displayed a construct length of 1,076 bp. Figures 1 and 2 document the saved cassette; the schematic export does not label its individual parts, so the table above supplies their positions (Asimov, 2026).

Figure 1. Kernel editor showing the saved cassette in baig-growingcolours (Asimov, 2026).

Figure 2. Schematic exported by Kernel. The export lacks individual part labels; the table above supplies the coordinates (Asimov, 2026).

4. Optimization and sequence checks

Kernel codon optimizer

I submitted the native 894 bp Tyr1 CDS to Kernel SIMPLE_0_3 with Escherichia coli as the expression host because K. rhaeticus was absent from its four available hosts. The result is 897 bp, with 172 substitutions across the 891 sense-coding bases. GC content changed from 43.85% to 44.93%, and the output added a TAA after the native TGA. Local translation confirmed the same 297 amino acids. I used this E. coli variant only as a comparison; I did not substitute it into the K. rhaeticus construct (Asimov, 2026).

Figure 3. Kernel optimizer output and recorded run settings (Asimov, 2026).

Sequence identity

I compared the saved CDS base for base with the published MT346028 Tyr1 region used to create it: all 894 positions match, and the translated protein matches QLI61466.1 (NCBI, n.d.). This is a comparison with the selected reference sequence, not a broad similarity search. The revised document reports no biosecurity scan result.

5. From digital design to DNA

For one prototype, I would order an insert covering the 1,076 bp cassette and place it in the verified pSEVA331Bb multiple cloning site using overlap-based assembly. I would then sequence the full insert and both vector junctions in an isolated clone before testing pigment formation. Overlap-based assembly has been demonstrated for joining DNA fragments, and sequence verification is important because synthesized DNA can contain errors (Carr et al., 2004; Gibson et al., 2009; Kosuri et al., 2010).

I believe one verified clone is enough to begin promoter and pigment tests. The assembly, sequencing and pigment tests remain proposed work.

6. Main unknowns and next tests

References

Addgene. (n.d.). pSEVA331Bb (Plasmid #78269). https://www.addgene.org/78269/

Asimov. (2026). Kernel [Software]. https://www.asimov.com/kernel

Baig, Z. (2026). Week 1 HW: Final project ideas. HTGAA Pages. https://pages.htgaa.org/2026b/zia-baig/week-01-hw-project-ideas/

Carr, P. A., Park, J. S., Lee, Y.-J., Yu, T., Zhang, S., & Jacobson, J. M. (2004). Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Research, 32(20), e162. https://doi.org/10.1093/nar/gnh160

Florea, M., Hagemann, H., Santosa, G., Abbott, J., Micklem, C. N., Spencer-Milnes, X., de Arroyo Garcia, L., Paschou, D., Lazenbatt, C., Kong, D., Chughtai, H., Jensen, K., Freemont, P. S., Kitney, R., Reeve, B., & Ellis, T. (2016). Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proceedings of the National Academy of Sciences, 113(24), E3431–E3440. https://doi.org/10.1073/pnas.1522985113

Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., III, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6, 343–345. https://doi.org/10.1038/nmeth.1318

iGEM Registry of Standard Biological Parts. (n.d.-a). BBa_B0015. https://registry.igem.org/parts/BBa_B0015

iGEM Registry of Standard Biological Parts. (n.d.-b). BBa_B0034. https://registry.igem.org/parts/BBa_B0034

iGEM Registry of Standard Biological Parts. (n.d.-c). BBa_J23104. https://registry.igem.org/parts/BBa_J23104

Kosuri, S., Eroshenko, N., LeProust, E. M., Super, M., Way, J., Li, J. B., & Church, G. M. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28, 1295–1299. https://doi.org/10.1038/nbt.1716

National Center for Biotechnology Information. (n.d.). Cloning vector pJV298-Tyr1, complete sequence (GenBank accession MT346028) [GenBank record]. https://www.ncbi.nlm.nih.gov/nuccore/MT346028

Walker, K. T., Li, I. S., Keane, J., Goosens, V. J., Song, W., Lee, K.-Y., & Ellis, T. (2025). Self-pigmenting textiles grown from cellulose-producing bacteria with engineered tyrosinase expression. Nature Biotechnology, 43(3), 345–354. https://doi.org/10.1038/s41587-024-02194-3