Week 2 HW: DNA Design

Repository name : Farrel Alfaza Marsetyo

Part 1: Define your expression target

I’m expressing a polypeptide enzyme, for this project I’m planning to express one specific enzyme for one specific disease, but in the future I plan to expand it to a class of similar enzymes that targets a number of rare diseases. The specific enzyme I’m planning to express is imiglucerase (brand name Cerezyme)

2. 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.

>imiglucerace keggD03020	
ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQAN
HTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNI
IRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLAS
PWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEP
SAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVV
LTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRL
GSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYK
QPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTI
KDPAVGFLETISPGYSIHTYLWHRQ

Imiglucerase enzyme obtained from kegg (link: https://www.kegg.jp/entry/D03020)

2. 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 chassis is CHO (chinese hamster ovary) cell line

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

As this enzyme is supposed to be injected cleanly (as naked protein molecule) into the bloodstream intravenously, therefore to aid the expression and purification process it is better for the enzyme to be secreted into the extracellular space. Some other reasons (courtesy of Claude)

1. Post-translational processing requires the secretory pathway. GBA needs N-linked glycosylation (which happens in the ER and Golgi) to get those terminal mannose residues that let macrophages take it up via the mannose receptor. Cytoplasmic expression bypasses the ER/Golgi entirely, so you’d get an unglycosylated, non-functional-for-therapeutic-purposes protein.
2. Proper folding and disulfide bonds. Your sequence has multiple cysteines (I count several C’s scattered through it) that likely form disulfide bonds. This chemistry happens in the oxidizing environment of the ER, not the reducing environment of the cytoplasm. Cytoplasmic expression would likely give you a misfolded, insoluble, or aggregated protein.
3. Purification logistics. If secreted into the CHO culture medium, you harvest by simply collecting the supernatant and purifying from there (chromatography steps). Cytoplasmic expression means you’d need to lyse cells, and you’d have to purify away from thousands of intracellular host cell proteins, which is messier and lower yield for a protein like this.
4. This is literally how Cerezyme/imiglucerase is made industrially — CHO cells secrete the recombinant enzyme into the medium, which is then purified through multiple chromatography steps before formulation.

Part 2: Design your genetic parts

Part 3: Assemble your construct

Expression construct for Imiglucerase constitutive expression on CHO cells. The construct consists of an imiglucerase CDS expressed under a CMV promoter with bGH poly(A) signal terminator and an N-terminus IgG kappa secretion tag from Mus musculus.

Future considerations : I’m planning to be able to swap the CDS easily in the future, so I’m considering on adding a BsaI or BbsI cut site with a BioBrick prefix to the CDS part so that it could be easily swapped with a simple GoldenGate reaction. Inspired by iGEM Gifu 2023

Part 4: Optimize and quality-check

Codon-optimize your CDS for your chosen host. Compare before and after: what changed, and why does codon usage matter for expression in this organism? Note any constraints you applied (e.g. avoiding certain restriction sites, GC content).

Asimov tools are used codon optimize and create CDS from kegg.jp. Protein sequences are put into the tool and settings are set for CHO cells. Stop codons are set for TAA and TGA. Constraints are automatically applied by the system, excluding BsaI, BsmbI, PaqCI, and BbsI, which I assumed is in adherence to the RFC1000 standard (GoldenGate/Loop) and the GoldenBraid assembly standard.

Search your sequence (BLAST / sequence search) against known sequences. Is your CDS what you think it is? Is it identical to a natural gene, or novel? What is the closest match?

BLAST results shows 100% hit to a few top targets. The CDS is identical to Sorex araneus glucosylceramidase beta 1 (GBA1) transcript variants X1 and X2 mRNA. It is also identical to Human glucocerebrosidase mRNA, complete cds. This result is expected since the enzyme should also be naturally expressed in human.

Run the safety screen on your construct. Summarize what it checks for and what it reports. If it flags something, explain whether it is expected for your design and how you would respond.

Safety screen was done using the SecureDNA tool on asimov. The safety screening shows no positive hit and showed that the sequence is safe to use.

Part 5: From design to build

How would you build it? Would you order it as synthetic DNA, assemble it from smaller fragments (e.g. Gibson assembly), or build it from a pool of oligos synthesized on a chip? Justify your choice given your construct’s length and complexity. What does synthesis constrain? Synthesis has error rates and struggles with certain sequences. Does your design contain anything hard to synthesize? How would error correction or fragment-based assembly change your approach? Prototype vs. scale. Mirroring HW1: the way you’d build one construct to test your idea may differ from how you’d produce DNA at the scale your product needs. Briefly describe both if they differ.

I plan to assemble it from smaller fragments in the modular spirit of synthetic biology. This is because I’m planning to make the expression system modular so that it could synthesize more than my current protein proof-of-concept (imiglucerase). Other considerations to think of:

1. Using modular parts means that when the system needs to be optimized, changing the parts is as simple as running another GoldenGate/GoldenBraid reaction in a tube and a thermocycler instead of designing multiple primers as in Gibson Assembly
2. The shorter length of my total construct makes this approach much more sensible since shorter fragments are more amenable towards a digest-ligate approach
3. Modularity means that the platform is easily expanded and compounded by other researchers and firm which would increase its scalability.

Synthesis constraints : so far I’m not aware of any synthesis contraints present on my sequence, a quick brainstorm with claude shows these general constraints in gene synthesis

**Sequence composition issues**

1. **High/low GC content.** Regions above ~65% GC or below ~30% GC are harder to synthesize accurately. High-GC regions are prone to incomplete deprotection/coupling during oligo synthesis and to secondary structure; low-GC regions can have weak, unstable duplexes during assembly.
2. **Homopolymer runs.** Long stretches of a single base (e.g., poly-A, poly-T, poly-G >8-10 bases) cause slippage errors during synthesis and sequencing verification.
3. **Repetitive sequences.** Tandem repeats, direct repeats, or near-identical sequences at a distance can cause mis-annealing during PCR-based assembly steps, leading to deletions or recombination between repeats.
4. **Secondary structure.** Sequences that fold into hairpins, stem-loops, or cruciforms (common in high-GC or palindromic regions) block polymerase progression during assembly and PCR amplification.

**Structural/genetic elements**

5. **Palindromic sequences,** especially long inverted repeats, can form hairpins that stall synthesis or cause the sequence to be unstable once cloned into a plasmid (bacteria may actively delete or recombine them).
6. **Restriction sites you didn't want,** if your assembly method uses Type IIS enzymes (Golden Gate, etc.) — internal cut sites for your chosen enzyme will fragment the sequence unexpectedly. This is a design problem more than a synthesis-difficulty one, but it can hold up a build.
7. **Toxic or growth-inhibitory sequences to the host** (usually _E. coli_ during cloning/propagation) — some ORFs, promoters, or membrane-disrupting peptides are toxic when expressed even at leaky basal levels, causing plasmid instability, mutation, or clone dropout during the bacterial amplification step that follows chemical synthesis.

**Practical vendor thresholds**

8. **Sequence length** — very long constructs (multi-kb) require more assembly/ligation steps, each with failure probability, compounding difficulty.
9. **Uniqueness/junction design** in multi-fragment assembly — poor junction sequences between fragments (e.g., unintended homology) can cause mis-assembly.

A cursory check on my construct shows that the whole sequence has a GC content of 51.29% which falls neatly within the ~30-59% constraint. However, an online GC content checker tool detected a potentilal CpG island in base 611-824, with a GC content of 57.9%. The GC content is still within the acceptable limits, however, downstream expression in CHO cells could potentially face problems, are CpG island are prone to methylation and silencing. Further changes are needed to improve this part of the sequence.

DNA Synthesis : synthesizing DNAs needed for industrial-level drug production is impossible with current synthesizers. However, using cell-based replicating mechanism is quite more economical compared to synthesizers. Therefore, to address the amount of DNA Needed, I’m planning on puting the construct on two backbones:

1. Backbone 1 : the main backbone, for expression in CHO cells.
2. Backbone 2 : replication backbone, for expression and replication in E. coli

E. coli cells such as DH5-alpha who had been selected from transformation efficiency or …, selected for DNA replication are perfect for this application. This is also in line with the modular principle of the sequence build philosphy since it could be easily swapped between backbones inside the GoldenGate/GoldenBraid system.

Part 6: What don't you know yet?

How to address this gap : read out on some parts used in CHO cell expressions, possibly from the iGEM Registry or other websites. Also read on the Gingko RAC specification, especially how they handle mammalian cells. Additionally, I need to run the numbers on CHO cells production of biologics.