Week 2 Pre Questions

I have answered the following questions to prepare for the lecture on DNA design and synthesis. These answers are based on the slides provided by Professor Jacobson, Dr. LeProust, and Professor Church.

Question,1

Natures machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome. How does biology deal with that discrepancy?

My Answer: Polymerase is very accurate but it still makes mistakes. The error rate is roughly one in ten to the power of nine to ten to the power of ten per base pair. Since the human genome is about three times ten to the power of nine base pairs long, mistakes would happen every time a cell divides if there were no corrections. Biology deals with this discrepancy by using proofreading and mismatch repair systems to find and fix errors during and after the copying process.

Question,2

How many different ways are there to code DNA nucleotide code for an average human protein? In practice what are some of the reasons that all of these different codes dont work to code for the protein of interest?

My Answer: There are many different ways to code for a single protein because the genetic code has redundancy. For an average human protein, the number of combinations can be more than ten to the power of one hundred. In practice, many of these codes do not work because of codon usage bias where certain cells prefer specific codes. Other reasons include mRNA secondary structures that block the process or sequences that are just too difficult to synthesize.

Question,3

Whats the most commonly used method for oligo synthesis currently? Why is it difficult to make oligos longer than 200nt via direct synthesis? Why cant you make a 2000bp gene via direct oligo synthesis?

My Answer: The most common method today is the phosphoramidite method. It is hard to make sequences longer than 200 bases through direct synthesis because of. the coupling efficiency. Even with a success rate of 99.5 percent at each step, the total yield for a 200 base sequence drops to about 36 percent. For a 2000 base gene, the success rate becomes effectively zero. This is why we must assemble long genes from smaller pieces instead of trying to make them all at once.

Question,4

What are the 10 essential amino acids in all animals and how does this affect your view of the Lysine Contingency?

My Answer: The ten essential amino acids are Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, and Lysine. Learning this makes me realize that the Lysine Contingency used in Jurassic Park is not a very strong safety method. Since lysine is found in many natural foods and plants, an organism could survive by finding it in the environment. This shows that we need to use synthetic amino acids that do not exist in nature to truly contain an organism.

Week 1 HW: Principles and Practices

ARM-Net: Alzheimer’s Recovery Micro-TNT Network

1.Biological Engineering Application

I am developing ARM-Net, a synthetic biology tool designed to overexpress CCT4 specifically in microglia. Unlike the full TRiC/CCT complex, CCT4　can function independently or as homo-oligomers to promote microtubule synthesis. By boosting these chaperonins, I aim to induce the formation of Tunneling Nanotubes (TNTs) between microglia, creating an “Intercellular Care Network” (ICN)

Source: Nuntaprut et al. (2024), “Microglia rescue neurons from aggregate-induced neuronal dysfunction,” Science.

Why: Current Alzheimer’s Disease (AD) treatments focus on slowing progression. ARM-Net aims for recovery. By networking microglia, we can distribute the overwhelming proteotoxic load (Amyloid-β,Tau) across a cellular collective for efficient degradation and allow healthy cells to “rescue” damaged ones by transferring mitochondria and resources via TNTs.

2. Governance and Policy Goals

Primary Goal: Ensuring Non-malfeasance and Biological Integrity

•The overarching goal is to ensure that the induction of TNTs (Tunneling Nanotubes) via CCT4 overexpression promotes brain recovery without introducing new pathological risks or irreversible side effects.

Sub-goal A: Prevention of Pathological Propagation

While TNTs are designed to facilitate the clearance of toxic proteins like Amyloid-β and Tau, they must not inadvertently act as a conduit for the spread of these very same pathogens or viral vectors across the brain.

•Actionable Metric:

Implement molecular “checkpoints” or filtering mechanisms within the TNT structure to ensure one-way or cargo-specific transport.

•Risk Mitigation:

Preventing spread of neurodegenerative diseases through the engineered network.

Sub-goal B: Safeguarding Cognitive Identity and Reversibility

Ensuring that the physical modification of the brain’s immune network does not alter the patient’s fundamental personality, memories, or self.

•Actionable Metric:

Integration of an inducible kill-switch or degradation domain that can dissolve the TNT network if behavioral side effects are observed.

•Ethical Standard:

Prioritizing the preservation of the brain’s natural cell death cycles. The tool must avoid creating a permanent “hyper-connected state” to maintain the ecological homeostasis of the neural environment.

3. Governance Actions

4.Scoring Table

5. Prioritization and Recommendation

Priority 1: Elimination of Pathogenic Propagation Risks

The absolute highest priority is ensuring that TNTs do not facilitate the spread of viruses or neurotoxic proteins. Unless this fundamental danger is completely eliminated through rigorous design, this project cannot even reach the starting line of clinical development. We must guarantee that our engineered “care network” does not inadvertently become a “superhighway” for the very pathology it aims to treat.

Priority 2: Maintenance of Biological Homeostasis

Clearing the requirement for systemic homeostasis is also a paramount necessity. The brain is a delicate ecosystem; any intervention that induces hyper-connectivity must not disrupt the natural metabolic balance, synaptic pruning, or the healthy turnover of cells.

Recommendation

prioritize a combination of Action 1 (Molecular Filtering) and Action 2 (Pulsatile Activation) as the primary strategy to address these critical hurdles.

•Technical Strategy:

By engineering CCT4 with cargo-specific filtering domains, we provide a physical barrier against pathogen spread.

•Regulatory Standard:

I recommend that national health agencies and the International Neuroethics Society mandate “Temporal Reversibility” as a non-negotiable standard. By ensuring the network is only active in “pulses,” we allow the brain to return to its natural homeostatic state between treatment cycles.

Week 2: DNA Design and Characterization

This week, I focused on the design, simulation, and cloning workflow for CCT4, the core component of my project, ICN (Intercellular Care Network).

1. Part 1: Benchling and In Silico Gel Art

Benchling Simulation

Having long been interested in Benchling, I used this assignment to dive deep into the tool. Instead of the suggested Lambda DNA, I worked with the CCT4 protein sequence relevant to my project. I simulated restriction digests using the following enzymes:

• Enzymes used: EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI, SalI

Gel Art: The Tree🌲

I used the virtual electrophoresis simulator to create “Gel Art” representing a tree. Initially, I struggled with the manual placement of bands, but once I understood the physical mechanism of fragment migration, I was able to dial in the design more effectively.

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2. Part 2: Wet Lab

As I do not currently have access to a wet lab facility, I focused on digital simulations and perfecting the genetic design for future implementation.

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3. Part 3: DNA Design Challenge - CCT4

Why CCT4? (Hypothesis)

CCT4 is a chaperonin that, when overexpressed, not only promotes the formation of TNTs (Tunneling Nanotubes) but also accelerates the speed of cargo transport through them. My hypothesis is that by overexpressing CCT4 in microglia, we can significantly speed up the clearance of toxic proteins like Tau, which are central to Alzheimer’s disease progression. (See my Week 1 HW for more details).

Sequence & Codon Optimization

Based on UniProt P50991 (Isoform 2), I used the IDT Codon Optimization Tool to design a DNA sequence optimized for expression in Escherichia coli (E. coli). Amino Acid Sequence: MPENVAPRSGATAGAAGGRGKGAYQDRDKPAQIRFSNISAAKAVADAIRTSLGPKGMDKM IQDGKGDVTITNDGATILKQMQVLHPAARMLVELSKAQDIEAGDGTTSVVIIAGSLLDSC TKLLQKGIHPTIISESFQKALEKGIEILTDMSRPVELSDRETLLNSATTSLNSKVVSQYS SLLSPMSVNAVMKVIDPATATSVDLRDIKIVKKLGGTIDDCELVEGLVLTQKVSNSGITR VEKAKIGLIQFCLSAPKTDMDNQIVVSDYAQMDRVLREERAYILNLVKQIKKTGCNVLLI QKSILRDALSDLALHFLNKMKIMVIKDIEREDIEFICKTIGTKPVAHIDQFTADMLGSAE LAEEVNLNGSGKLLKITGCASPGKTVTIVVRGSNKLVIEEAERSIHDALCVIRCLVKKRA LIAGGGAPEIELALRLTEYSRTLSGMESYCVRAFADAMEVIPSTLAENAGLNPISTVTEL RNRHAQGEKTAGINVRKGGISNILEELVVQPLLVSVSALTLATETVRSILKIDDVVNTR

3.4. Specific Procedures in Cell-Based Expression (E. coli Expression System)

For this project, I am assuming the process using E. coli, which is the most common and reliable cell-based expression system.

1. Transformation Introduce the designed DNA (plasmid) into the E. coli cells. This is the process of having the E. coli read the CCT4 blueprint by applying stimuli such as heat shock.

2. Culturing and Expansion Efficiently grow only the E. coli that harbor the DNA in a medium containing antibiotics. We maximize the population of E. coli in an incubator, providing optimal temperature and agitation.

3. Induction of Expression Once the E. coli has sufficiently proliferated, reagents like IPTG are added to flip the CCT4 protein production switch. Protein synthesis within the cells begins immediately from this point.

4. Recovery and Purification Once a sufficient amount of protein has been produced, the E. coli cell walls are disrupted (lysis) to retrieve the contents. Subsequently, tags such as His-tags are used to extract only the pure CCT4 protein from the mixture.

3. Reasons for Choosing This Method

While cell-based methods take more time compared to cell-free methods, their greatest advantage lies in the ability to mass-produce large quantities of protein at a low cost. In order to broadly deploy the ICN network and care for many cells in the future, this E. coli-based mass production system will be an indispensable infrastructure.

4. Part 4: DNA Synthesis & Cloning

I constructed a CCT4 expression cassette for E. coli transformation.

• Design Refinement: I resolved initial issues by adding a Stop Codon and a His-tag for purification. I excluded promoters/RBS as they are provided by the destination vector.

• Execution: I exported the FASTA from Benchling and uploaded it to Twist Bioscience. After cloning into the pTwist Amp vector, I imported the Genbank file back into Benchling to finalize the plasmid map. This was my first-ever digital cloning experiment!

5. Part 5: DNA Reading, Writing, and Editing

5.1 DNA Reading

• Target DNA and Reason: I will target the CCT4 gene region within human microglial cells and its corresponding mRNA to reflect its expression status. The objective is to quantitatively understand how chaperonin function within microglia changes during the progression of Alzheimer’s disease and to verify if the engineered genes are functioning as intended.

• Technology and Reason: I will utilize Nanopore Sequencing, which is classified as a 3rd-generation technology. Unlike conventional methods, it can read long-read DNA or RNA in real-time without PCR amplification. This provides a significant advantage by allowing analysis without losing structural complexity or epigenetic information related to repair networks.

• Detailed Process:

  • Input: High-purity genomic DNA extracted from microglia.
  • Preparation: The extracted DNA is purified to an appropriate length, and adapters containing motor proteins are ligated to both ends to guide the DNA into the nanopores.
  • Decoding: As the DNA passes through the nanopore, changes in ionic current are detected as waveforms, which are then converted into base sequences using neural networks (basecalling).
  • Output: Data in FASTQ format, containing base sequences and quality scores.

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5.2 DNA Writing

• Synthetic DNA and Reason: I will synthesize an optimized sequence of the human CCT4 gene designed for maximum production efficiency in E. coli. This physical DNA fragment is required to create a prototype aimed at enhancing microglial intercellular care functions and suppressing the aggregation of abnormal proteins.

• Technology and Reason: I will use the Silicon-based Phosphoramidite method. This technology, utilized by companies like Twist Bioscience, enables the simultaneous synthesis of tens of thousands of DNA fragments on a microscopic silicon plate, achieving overwhelming scalability and cost reduction for individual gene synthesis.

• Detailed Process:

  • Key Steps: Each cycle consists of four chemical reactions—deprotection, coupling, oxidation, and capping—to accurately assemble the sequence base-by-base.
  • Limitations: Due to the nature of the chemical synthesis process, a length of approximately 1.6 kb requires a lead time of about two weeks. Additionally, since error rates accumulate as the sequence lengthens, post-synthesis verification via sequencing is mandatory.

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5.3 DNA Editing

• Target DNA and Reason: I will target safe harbor loci, such as the AAVS1 region within the microglial cell genome. This allows for the stable integration of the exogenous CCT4 gene into the genome to maintain long-term therapeutic effects while avoiding the risk of disrupting other vital genes.

• Technology and Reason: I will use CRISPR/Cas9. This technology allows for extremely high-precision genome editing, as designing guide RNAs (gRNA) to target specific base sequences is straightforward.

• Detailed Process:

  • Editing Mechanism: A complex consisting of gRNA, which binds to the target genomic sequence, and the Cas9 enzyme, which acts as molecular scissors, is introduced into the cell. After the DNA is cleaved at the target site, accurate gene insertion is performed via Homology-Directed Repair (HDR) using the provided CCT4 template.
  • Preparation and Input: This requires selecting the target sequence, designing the gRNA, and preparing the Cas9 enzyme along with the plasmid containing the CCT4 gene to be integrated.
  • Limitations: There are risks of off-target effects, where unintended locations are edited, and physical constraints such as the tendency for gene integration efficiency to decrease in non-dividing or slowly dividing cells like microglia.

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project