Week 2 HW: DNA Design

HTBAA Homework #2

Due: Tuesday September 29, by 2:30p ET (delayed due to Asimov access) Discussion Monday, October 5

Part 0: Set up in Kernel

This construct can be found in repository: odim-project in asimov.

Part 1: Define your expression target

Start from the project you proposed in Homework #1 and pin down exactly what your cells need to make. Answer in your write-up:

What molecule are you expressing? A protein, an RNA, or an enzyme (or small set of enzymes) for a pathway. If your HW1 product is a material, food, or chemical, identify the protein(s) that produce it and design for those.

The first step in the process is engineering an amylolytic industrial yeast strain expressing α-amylase and glucoamylase genes. This HW will focus on that step, which is what will generate the sugars needed for fermentation into bacterial cellulose.

Reference (researched using Claude):

What is the sequence? Give the protein or RNA sequence, or the specific gene, you will encode, and say where it comes from (a database accession such as UniProt or NCBI, a known part, or de novo). Cite it.

Reference (researched using Claude):

What host or chassis? The living system from HW1, for example E. coli, S. cerevisiae, CHO or another mammalian line, or a cell-free system. Your host drives almost every part choice that follows.

The cited paper uses industrial diploid Saccharomyces cerevisiae, but I’m doing this in a lab version of S. cerevisiae.

Where does the product need to go? Cytoplasm, secreted, membrane, or a compartment? This decides whether you need a signal peptide or targeting sequence.

Secreted enzymes

Part 2: Design your genetic parts

This construct is built from the below parts.

Transcription start – promoter: GAP & TEF1

For the promoters, I used the same promoters cited in the paper above, but paired them with an enzyme arbitrarily:

  • Sequence 1 -> TEF1 promoter (by BioClub Tokyo)
  • Sequence 2 -> BBa_K530008 = GAP (also called TDH3) promoter

Translation start & Coding sequence – CDS: α-amylase & glucoamylase

These were the genes I could find to represent α-amylase & glucoamylase, and both had translation folded within them:

  • Sequence 1 -> R. emersonii CDS from KT246498.1 (α-amylase) – carries start/stop and signal peptide within it
  • Sequence 2 -> BBa_K4845008 (glucoamylase) – GenBank MW082635, yeast codon-optimized, carries start/stop and signal peptide within it. This is a slightly different source than I was initially looking for, but matches in sequence.

Transcription stop – Terminator:

Similarly, I used the same terminators as the cited paper. I also checked them against standardized parts for S. cerevisiae:

  • Sequence 1 -> CYC1 (by BioClub Tokyo)
  • Sequence 2 -> BBa_J63002 (ADH1)

Reference (researched using Claude):

Vector context – backbone:

  • BBa_K5007025 (pRS426 derivative that acts as pRS425) – provides 2µ origin, LEU2, and builds in E. coli.

Part 3: Assemble your construct

image image pRS425 plasmid TEF1 R. emersonii α-amylase CYC1 TDH3 glucoamylase ADH1

Part 4: Optimize and quality-check

Sequence Alignment: I found two different enzymes I thought were α-amylase and compared their sequences, which led me to understand they were actually different despite being labeled the same. I instead decided to import my own from https://www.ncbi.nlm.nih.gov/nuccore/KT246498.1. BLAST came back with the specific parts I started with for both enzymes.

Codon Optimizer: Glucoamylase is already codon optimized, so I tried α-amylase. It updated to use TTG, GCT, AGA, GGT, TCT, CCA like the glucoamylase strain.

Biosecurity Sequence Scanner: No flagged sequences were detected in the biosecurity scanner for security-relevant, controlled, or regulated sequences.

Part 5: From design to build

For the purposes of lab/testing, I would order α-amylase and glucoamylase as separate fragments to aid in synthesis. Because it combines two enzymes/functions it is a longer sequence to synthesize, and can benefit from this approach (Gibson et al. 2009).

I’d want to ensure the set up can produce the amount of glucose needed outside the cell. Ordering these fragments can help derisk the process, before synthesizing a version that can be scaled up with appropriate error correction (Kosuri et al. 2010, Tian et al. 2004).

Part 6: What don’t you know yet?

As in Homework #1, close by naming the edges of your design. Identify the 2–3 biggest unknowns or risks in your construct. For example: Will the promoter give the right expression level? Will the protein fold and be active in this host? Will it be secreted? Is codon optimization enough, or do you need to tune it empirically?

For each, state how you would find out: the measurement, experiment, or design variant you would try next. A good answer here shows you know which decisions are still bets.

I chose promoter/terminator pairs for each enzyme arbitrarily, but in the cited paper they use both with each. I would want to see the impact of this selection, test the otherway around, and maybe even test 2 pairs of each enzyme with each set of promoter/terminators. I would measure glucose production.

I don’t know what the actual amount of glucose will be produced and how the design might increase that/ensure it is produced outside of the cell. As mentioned in the last section, my first step would be to order the enzymes and try to produce enough glucose, then attempt to synthesize a matching amount, before setting up the system to scale.