Week 2 HW: DNA DESIGN

Part 1 — Define your expression target What molecule are you expressing? For this assignment, I would focus on expressing ACC deaminase, an enzyme encoded by the acdS gene. My larger project from Homework #1 was a microbial inoculant for corn, beans, and squash that could potentially help the plants with nutrient availability and environmental stresses such as drought and flooding. A complete inoculant would probably involve several microorganisms and biological functions, which would be too complicated to design as one genetic construct. For this homework, I am therefore focusing on one possible part of that system. ACC deaminase breaks down ACC (1-aminocyclopropane-1-carboxylate), which is involved in the plant’s production of the stress-related hormone ethylene. My idea is that a plant-associated bacterium expressing ACC deaminase could potentially help moderate plant stress responses. I do not expect this single enzyme to make corn, beans, or squash drought- or flood-resistant by itself. Instead, it would be one possible function in the larger microbial community proposed in Homework #1. What is the sequence? I would use the acdS gene encoding ACC deaminase from Pseudomonas putida UW4. The protein is 338 amino acids long, and its reviewed UniProt accession is Q5PWZ8. The corresponding genomic sequence is associated with GenBank accession AF047710. UniProt For Kernel, I would import the natural acdS sequence and then use Kernel’s codon-optimization tool for my chosen host rather than trying to design the protein myself. What host or chassis? For the assignment, I would use Pseudomonas putida as the bacterial host. I chose a bacterial host because the final product from Homework #1 is supposed to be a microbial soil/root inoculant. Pseudomonas species are common in plant-associated microbial research, and the original acdS gene I selected already comes from a P. putida strain. This also keeps my first genetic design relatively simple rather than trying to engineer the plant itself. Where does the product need to go? I would keep ACC deaminase inside the bacterial cell (cytoplasm). Because of this, I would not initially add a secretion signal peptide. The bacteria would colonize the area around or near plant roots and express the enzyme themselves.

Part 2 — Design the genetic parts This is where I’d keep your design intentionally basic. The assignment’s table on page 2 says that a prokaryotic design needs a promoter → RBS → coding sequence → terminator, along with a suitable vector backbone. HTBAA Homework #2_ DNA Design Your preliminary parts could therefore be: Part What I would use Why Promoter Constitutive bacterial promoter available in Kernel Keeps acdS expression relatively simple for this first design. RBS Standard bacterial RBS available in Kernel Allows the bacterial ribosome to begin translating the protein. CDS acdS, based on P. putida UW4 Q5PWZ8 Produces the ACC deaminase enzyme that is the focus of my design. Signal peptide None I currently intend the enzyme to remain inside the bacterial cell. Terminator Standard bacterial terminator available in Kernel Provides a clear end to transcription. Backbone Pseudomonas-compatible/broad-host-range plasmid available in Kernel Allows the construct to be maintained in the bacterial host.

I wouldn’t invent specific promoter/backbone names yet. Look at what’s actually available in your Kernel workspace first. The homework specifically allows you to reuse an existing backbone. HTBAA Homework #2_ DNA Design Once you show me what Kernel offers, we can choose among the real parts rather than making your paper sound more knowledgeable than you actually were when doing the exercise. Part 3 — Assemble the construct My basic construct would be: Promoter → RBS → acdS CDS → Terminator inside a Pseudomonas-compatible plasmid backbone. I would check in Kernel that all of the parts face the same direction, that the coding sequence has the expected start and stop codons, and that there are no unexpected stop codons within the gene. The purpose of this construct would be to make the bacterial host produce ACC deaminase. This represents only one possible stress-related component of the larger microbial inoculant I proposed in Homework #1. You’ll then insert the Kernel schematic/screenshot here, because that’s explicitly part of the deliverable. HTBAA Homework #2_ DNA Design Part 4 — Optimize and quality-check We can’t honestly finish this section until you actually run Kernel because the assignment asks what each tool returned. HTBAA Homework #2_ DNA Design Once you run them, your responses can be simple: Codon optimization: I used Kernel to codon-optimize the acdS coding sequence for Pseudomonas putida. The amino acid sequence should remain the same, but some DNA codons were changed to ones better suited to the host. [ADD YOUR KERNEL RESULTS: number/percentage of bases changed, GC changes, etc.] Sequence search/BLAST: I searched the coding sequence against known sequences. The closest match was [RESULT]. This was [expected/unexpected] because my sequence was based on the naturally occurring acdS gene from P. putida UW4. Safety screen: I ran Kernel’s safety screen on the finished construct. The tool checks the sequence for [COPY/SUMMARIZE WHAT KERNEL SAYS IT CHECKS]. My construct [did/did not] produce a warning. [EXPLAIN RESULT.] Don’t fabricate those results before running it. Part 5 — From design to build For a first prototype, I would probably order the acdS coding sequence as synthetic DNA and assemble it into an existing plasmid backbone, rather than trying to synthesize an entire plasmid from scratch. One possible assembly method would be Gibson assembly. Gibson et al. demonstrated that DNA fragments with overlapping ends can be assembled together in a single reaction, including much larger DNA molecules than the construct I am proposing. This would allow the synthetic acdS sequence to be joined with an existing vector containing the other necessary parts. DNA synthesis is not perfect, however. Errors can occur during synthesis, particularly as DNA designs become longer or more complicated. Carr et al. demonstrated methods for detecting and correcting errors in synthetic DNA. Therefore, after assembling my construct, I would want the finished sequence to be checked before assuming that it exactly matches my computer design. For an early experiment I would only need a small number of DNA constructs to test different versions of the design. If this eventually became an agricultural product, however, I would not need to manufacture a new plasmid for every plant or every dose of inoculant. Once the desired bacterial strain had the genetic construct, the bacteria themselves would be grown in larger quantities. The DNA-design stage is therefore mainly important for creating and testing the microbial strain rather than producing each final dose of inoculant. Those two assigned papers are particularly appropriate because the homework explicitly asks you to ground this section in at least two readings, and both Gibson assembly and DNA-synthesis error correction are included in the assigned reading list. HTBAA Homework #2_ DNA Design HTBAA Homework #2_ DNA Design Part 6 — What don’t you know yet?

1. Will producing more ACC deaminase actually improve stress tolerance in all three crops? Corn, beans, and squash may respond differently. I would test plants inoculated with the engineered bacteria against plants inoculated with the unmodified bacteria under normal, drought, and waterlogged conditions. Measurements could include survival, growth, root growth, and final biomass.
2. How much ACC deaminase should the bacteria produce? More expression is not automatically better. A very strong promoter could put unnecessary stress on the bacterium, while a weak promoter might not produce enough enzyme to affect the plant. I could compare constructs using promoters of different strengths and measure both bacterial growth and ACC-deaminase activity.
3. Would the engineered bacteria successfully interact with plants and other microorganisms in a real milpa? My original idea depends on a community of microorganisms interacting with three different crops. Even if this single engineered bacterium works in a controlled experiment, it may behave differently in actual soil. I would eventually test it first with each crop individually and then with corn, beans, and squash grown together.