Week 2 HW: DNA Design

HTBAA Homework 2

Part 1. Define Your Expression Target

Project objective

My project aims to develop a bacterial cellulose biofoam incorporating textile waste and explore its use in interior panels for thermal and acoustic insulation. For this assignment, I have narrowed the genetic design to an earlier question: can the organization and porosity of cellulose be modified while the bacterium produces it? Incorporating textile fibers is part of the biofabrication process. I do not assume that it requires a genetic modification or that more porous cellulose will automatically become an insulating foam.

What molecules am I expressing?

Cellulose is the final material, but it is not a protein that I can encode directly in a genetic construct. The bacterium produces it using its own synthesis machinery. Within that machinery, BcsA (catalytic protein that forms cellulose chains) participates in synthesis, while BcsD (protein associated with the organization of those chains) contributes to the structure of the material (Römling & Galperin, 2015).

In this assignment, I do not propose expressing an additional copy of BcsD. Instead, I propose expressing dCas9 (deactivated Cas9 protein that can bind to DNA without cutting it) and an sgRNA (single guide RNA that directs the protein to a specific DNA region). Together, they form a CRISPRi system (genetic interference system that reduces gene expression). My aim is to decrease the expression of a gene already present in the bacterium and observe whether this changes the structure of the cellulose it produces (Qi et al., 2013).

This choice is based on Huang et al. (2022), who reduced the expression of bcsD (gene associated with cellulose organization) in Gluconacetobacter xylinus CGMCC 2955 and observed changes in porosity estimated from microscopy images. Their result supports my research question, although it does not yet show that the same effect will occur in the strain selected for my project.

What is the sequence, and where does it come from?

The gene I want to regulate, rather than introduce, is present in the reference chromosome of Komagataeibacter rhaeticus iGEM, accession LT575493.1. In that record, it is annotated as acsD (gene for subunit D of the cellulose synthesis system), at positions 670250–670720, with the identifier KRIGEM_00627 and the product description “Cellulose synthase operon protein D” (European Nucleotide Archive, 2016). I use acsD when referring to the iGEM genome annotation and bcsD when referring to the gene studied by Huang et al. (2022).

As an initial candidate for the RNA unit, I selected the 337+ guide region described by Huang et al. (2022): 5′-CCGCTGGATCACGTCGCAGC-3′. Comparison with LT575493.1 shows that all 20 bases match positions 670588–670607 of acsD. This supports considering the region for a design in the iGEM strain; it does not establish how strongly it would reduce gene expression. The complete sgRNA sequence (single guide RNA, including its structural scaffold) and the specific dCas9 CDS (DNA coding sequence for the dCas9 protein) still need to be selected from verifiable sources before these parts can be recorded as final in Kernel.

What is the host or chassis?

The host, or chassis, will be Komagataeibacter rhaeticus iGEM, a cellulose-producing bacterium with a published genome and documented tools for modifying gene expression (Florea et al., 2016; Teh et al., 2019). Using this strain as my reference allows me to connect the proposal to a specific chromosomal sequence. It also means that the promoters, the sequences needed to produce dCas9, and the vector must be chosen with their function in this bacterium in mind. A guide region matching its genome does not, by itself, demonstrate that the complete system will work in this strain.

Where do the products need to go?

dCas9 (regulatory protein) and the sgRNA (single guide RNA) need to act inside the bacterium, where they can recognize the DNA containing acsD. For that reason, I do not propose adding a signal peptide to secrete them. BcsD (protein involved in cellulose organization) is part of the bacterium’s natural system; my design aims to change its expression level, not its cellular destination.

Bacterial cellulose, the material of interest, is produced outside the cell. At a later stage, I will study how this network grows in the presence of textile fibers and whether the resulting composite has the properties needed for an interior panel. The intracellular location of the regulatory system and the extracellular formation of the material therefore serve different functions within the same proposal.

Part 2. Design Your Genetic Parts

I propose organizing the system into two expression units. The first would produce dCas9 (regulatory protein without DNA-cutting activity) and would require a bacterial promoter, an RBS (ribosome binding site, a sequence that helps initiate protein production), a dCas9 CDS (DNA coding sequence for the protein), and a transcription terminator. The second would produce the sgRNA (single guide RNA): it would require a promoter, the guide sequence joined to its structural scaffold, and a terminator. This second unit does not need an RBS because its functional product is an RNA, not a protein. Together, dCas9 and the guide could obstruct transcription of the selected gene, which is the principle of CRISPRi (genetic interference system) (Qi et al., 2013).

I selected the region called 337+ by Huang et al. (2022) as my initial candidate. Its 20-base recognition sequence, 5′-CCGCTGGATCACGTCGCAGC-3′, appears without differences in the chromosome of K. rhaeticus iGEM, at positions 670588–670607 of the gene annotated as acsD. Comparison with the LT575493.1 FASTA file (text format containing a DNA sequence) showed a 20/20-base match and an adjacent NGG-type PAM (short DNA motif required for recognition by the corresponding version of Cas9). I also checked the other four regions published by Huang, and all matched exactly in this reference genome. I chose 337+ as an initial candidate because, in the published experiment, it was associated with relative bcsD expression of 29.39% compared with the control, one of the strongest reductions in that study. That figure describes the result in G. xylinus CGMCC 2955; it is not a quantitative prediction for the iGEM strain (Huang et al., 2022; European Nucleotide Archive, 2016).

To complete the construct in Kernel, I still need to select a specific dCas9 sequence, promoters, an RBS, terminators, and a vector that can be maintained and selected in K. rhaeticus iGEM. Teh et al. (2019) provide evidence of genetic parts for cellulose-producing bacteria and genetic interference in a related species, but this does not automatically validate a particular combination in my strain. In Kernel, I will record each part’s type, name, sequence, and verifiable source, and distinguish imported parts from my own design decisions. Until those sequences have been selected and checked, the diagram represents a proposed architecture, not a complete plasmid ready for synthesis.

My hypothesis is that reducing acsD expression will allow me to explore changes in the microstructure and porosity of cellulose produced by the iGEM strain. The sequence match makes it reasonable to study Huang’s guides in this strain, but the strength of regulation, bacterial growth, and material properties must be evaluated experimentally. Later, I can study whether that cellulose integrates with textile fibers and whether the resulting material has useful properties for an interior panel.

Part 3. Assemble Your Construct

To be developed.

Part 4. Optimize and Quality Check

To be developed.

Part 5. From Design to Build

To be developed.

Part 6. What Don’t You Know Yet?

To be developed.

Note on AI Use

I used ChatGPT (OpenAI, 2026) to review the literature, compare the sequences reported by Huang et al. with LT575493.1, and support the writing of Parts 1 and 2. I checked the claims against the cited article and records.

References

European Nucleotide Archive. (2016). Komagataeibacter rhaeticus strain iGEM genome assembly, chromosome 1 (record LT575493.1) [Data set]. EMBL-EBI. https://www.ebi.ac.uk/ena/browser/view/LT575493.1

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

Huang, L.-H., Li, X.-J., Sun, X.-W., Wang, X., Wang, Y.-T., Jia, S.-R., & Zhong, C. (2022). Regulating the structure of bacterial cellulose by altering the expression of bcsD using CRISPR/dCas9. Chinese Journal of Biotechnology, 38(2), 772–779. https://doi.org/10.13345/j.cjb.210318

Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173–1183. https://doi.org/10.1016/j.cell.2013.02.022

Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: Diversity of operons, subunits, products, and functions. Trends in Microbiology, 23(9), 545–557. https://doi.org/10.1016/j.tim.2015.05.005

Teh, M. Y., Ooi, K. H., Teo, S. X. D., Bin Mansoor, M. E., Lim, W. Z. S., & Tan, M. H. (2019). An expanded synthetic biology toolkit for gene expression control in Acetobacteraceae. ACS Synthetic Biology, 8(4), 708–723. https://doi.org/10.1021/acssynbio.8b00168