Week 2 Lecture Prep

PRE-LECTURE ABOUT CLASS 2

SLIDES ABOUT PROFESSOR JACOBSON

-Question 1 When biological polymerase copies DNA, it makes about 1 mistake per million base pairs (1:10^6).Since the human genome has around 3.2 billion base pairs, that error rate would mean every time one of my cells divides, it would introduce over 3,000 mistakes if there weren’t any correction mechanisms. There’s a 3’-5’ exonuclease that catches and removes errors during DNA synthesis, and then the MutS repair system acts as a backup to fix any mismatches that slipped through afterward. Together, these mechanisms keep my genetic information stable across cell divisions.

-Question 2 The average human protein is about 345 amino acids long (roughly 1036 base pairs of DNA). Because the genetic code is redundant, there are approximately 3^345 different DNA sequences that could produce the same protein—an astronomically large number. However, most of these sequences don’t actually work well in practice. Some fold into secondary structures like hairpins that block the copying machinery.

SLIDES ABOUT DR.LEPROUST -Question 1 The method that’s most commonly used today is called the phosphoramidite method, or phosphoramidite chemistry. From what I learned, this technique was developed back in 1981 by a researcher named Caruthers, and it’s still the gold standard in the industry. It’s used for both the traditional column-based synthesis approach and the newer silicon-based platforms that companies are working with now.

-Question 2 Even though each individual base might attach with really high efficiency, small errors happen at every single step of the process. By the time you reach 200 nucleotides, the percentage of molecules that are perfect and full-length has fallen to around 37% or even lower.

-Question 3 When I think about the cumulative error rate logic, the probability of building a perfect 2000 base pair molecule one base at a time is essentially zero. That’s why in practice, when scientists need to manufacture long genes, they don’t try to synthesize them directly. Instead, they make shorter verified fragments (oligos) and then assemble those pieces together to build the full-length gene.

SLIDES ABOUT DR.CHURCH There are 10 amino acids that humans can’t make on their own and have to get from food -> Phenylalanine (F), Valine (V), Threonine (T), Tryptophan (W), Isoleucine (I), Methionine (M), Histidine (H), Arginine (R), Leucine (L), and Lysine (K). The “Lysine Contingency” is a weak biocontainment strategy because lysine is already essential to all animals.

Week 1 HW: Principles and Practices

1. Biological engineering application: The development of various therapies in which they employ stem cells in Peru, to treat neurodegenerative diseases and chronic diseases. Along with this the appropriate regulations for this type of therapy. -> Why I chose this application: Because in Peru there is an increase in the development of various clinical therapies for diseases that have been very expensive to access, so the emergence of stem cell applications are recent. This is also due to a problem, this is more than anything focused on the little regulation on this type of treatment, which limits the creation of more trained centers, generating a delay in the access of new therapies and research.

2. Ethical future:

• Governance and policy objectives in Peru Overall objective: To establish a comprehensive framework that ensures stem cell therapies in Peru are safe, effective, and equitably accessible across all health sectors—both private and public—while fostering sustainable development of regenerative medicine in the country.

In addition, a specific and clear regulation must also be provided, which must be supervised by one or more public entities. Also to have control over the misleading commercial use of stem cells without clinical evidence, in order to provide health security for patients in the face of medical fraudulence. Finally, the approach of accessibility and equity, since what is expected to be achieved through the creation of centers with stem cell therapies is to facilitate access to these therapies in public and private hospitals, avoiding that they are only for sectors of high purchasing power those who have access to these therapies, this would help to reduce a gap in the health system that every year continues to be larger.

3. Governance Actions

4. Evaluation of Governance Actions

(Score from 1-3, with 1 as the best and 3 the least effective)

5. DRAW AND THE BEST OPTION

The best option→ Creation and implementation of a specific and explicit regulation focused on stem cell therapies.

Justification: It is essential to establish clear and precise regulations, in order to avoid risks for patients

Second option→ Development of a program for equitable access therapies in public hospitals.

Justification: Since stem cell therapies are expensive, there is a need to generate and promote greater investment and infrastructure development.

Third option → Sanctions for clinics that offer treatments without scientific support or violate patient confidentiality.

Justification: Seeking to prevent medical fraud and ensure that only viable and safe therapies are offered to patients across the country.

AUDIENCE :

This proposal is addressed to the Ministry of Health, which is responsible for implementing regulations, standards and properly supervising the correct application of cell therapies in Peru.

REFLECTION

When I think back on our class conversations about ethics and safety, one ethical issue really stuck with me: the idea of “Therapeutic Misconception” and how desperate patients’ hope can be exploited. Honestly, before doing this assignment, I always thought stem cell regulations were just red tape—like bureaucratic obstacles or technical checkboxes researchers had to deal with. But now I understand there’s something much deeper going on ethically. When people are vulnerable and desperately searching for treatments, especially when they’re running out of options, they can end up being sold therapies that haven’t actually been proven to work.

PRE-LECTURE ABOUT CLASS 2

SLIDES ABOUT PROFESSOR JACOBSON

-Question 1 When biological polymerase copies DNA, it makes about 1 mistake per million base pairs (1:10^6).Since the human genome has around 3.2 billion base pairs, that error rate would mean every time one of my cells divides, it would introduce over 3,000 mistakes if there weren’t any correction mechanisms. There’s a 3’-5’ exonuclease that catches and removes errors during DNA synthesis, and then the MutS repair system acts as a backup to fix any mismatches that slipped through afterward. Together, these mechanisms keep my genetic information stable across cell divisions.

-Question 2 The average human protein is about 345 amino acids long (roughly 1036 base pairs of DNA). Because the genetic code is redundant, there are approximately 3^345 different DNA sequences that could produce the same protein—an astronomically large number. However, most of these sequences don’t actually work well in practice. Some fold into secondary structures like hairpins that block the copying machinery.

SLIDES ABOUT DR.LEPROUST -Question 1 The method that’s most commonly used today is called the phosphoramidite method, or phosphoramidite chemistry. From what I learned, this technique was developed back in 1981 by a researcher named Caruthers, and it’s still the gold standard in the industry. It’s used for both the traditional column-based synthesis approach and the newer silicon-based platforms that companies are working with now.

-Question 2 Even though each individual base might attach with really high efficiency, small errors happen at every single step of the process. By the time you reach 200 nucleotides, the percentage of molecules that are perfect and full-length has fallen to around 37% or even lower.

-Question 3 When I think about the cumulative error rate logic, the probability of building a perfect 2000 base pair molecule one base at a time is essentially zero. That’s why in practice, when scientists need to manufacture long genes, they don’t try to synthesize them directly. Instead, they make shorter verified fragments (oligos) and then assemble those pieces together to build the full-length gene.

SLIDES ABOUT DR.CHURCH There are 10 amino acids that humans can’t make on their own and have to get from food -> Phenylalanine (F), Valine (V), Threonine (T), Tryptophan (W), Isoleucine (I), Methionine (M), Histidine (H), Arginine (R), Leucine (L), and Lysine (K). The “Lysine Contingency” is a weak biocontainment strategy because lysine is already essential to all animals.

Week 3 HW: OPENTRONS

Paper: Accelerated high-throughput imaging and phenotyping system for small organisms

• Link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0287739

This paper details the creation of a high-throughput experimentation (HTE) platform built around duckweeds — specifically Lemna minor, a tiny aquatic plant with applications in bioremediation and biofuel research. To run large-scale evolutionary ecology experiments, the team combined an Opentrons OT-2 liquid handling robot with a custom autonomous imaging system, creating a pipeline capable of operating at a scale that would be practically impossible by hand. The central engineering challenge was that standard liquid handling robots are designed to work with, unsurprisingly, liquids. Duckweeds are solid floating plant fronds, which meant the OT-2 needed to be rethought for a very different kind of material. The researchers solved this by replacing the standard pipette tips on the OT-2’s P300 pipette heads with commercial inoculation loops. These loops exploit capillary action to gently lift individual fronds from the water’s surface, allowing the robot to pick and place solid biological matter with the same reliability it would otherwise bring to liquid transfers. This seemingly simple hardware modification had enormous practical consequences. By enabling automated handling of the plants, the team was able to design an experiment encompassing 6,000 individual microcosms spread across 2,000 distinct combinations of nutrients and microbes — a scale of experimental complexity that manual pipetting and plant placement could never realistically achieve, given how tedious and error-prone working with tiny floating organisms at high volume would be for human researchers.

Week 2 HW: DNA READ, WRITE, & EDIT

DNA Design Challenge

• Protein: GFP (Green Fluorescent Protein)
• Reason: Because GFP is commonly used as a biological marker to visualize various cellular processes due to its green fluorescence.

sp|P42212|GFP_AEQVI Green fluorescent protein OS=Aequorea victoria OX=6100 GN=GFP PE=1 SV=1 MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL VTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

• Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence

GFP DNA >ATGTCCAAGGGTGAGGAGCTGTTTACCGGCGTGGTTCCGATTCTTGTGGAATTAGACGGCGATGTCAACGGCCACTTCTCCGTTTCT GGCGAGGGCGAGGGAGGCGACGCCACGTATGGCAAATTGACCCTGAAGTTTATTTGCACGACCGGAAAATTGCCTGTACCGTGGCCCACACTTTGGT CACTACCGTTATCAATGTTTCTCGCTATCCGGACCACATGAAGCAGCATGACTTCTTTAAAAGTGCAATGCCCGAGGGTTATGTTCAAGAGCGGACCA TCTTTTTTAAAAGACGACGGCAACTACAAGACGCGCGAGGTGAAGTTCGAGGGCGACACGCTGGTGAATCGGATTGAGTTAAAAGGAATTGACTTTAA AAGATGACGGCAACATCCTTGGACATAAGTTAGAGTACAATTATAATTCAAACCACGTGTACATCATGGCCGACAAACAAAAAAACGGCATCAAGGTA AACTTTAAAATTAGACATAATATCGAGGATGGCAGTGTTCAATTAGCCGACCATTACCAACAGAACACCGATAGGCGGACGGTCCTGTATTACCTGAC AACCATTACCTTAGCACGCAGTCTGCACTGTCCAAGGACCCAAATGAGAAACGAGGACCATATGGTGTTGCTAGAGTTCGTTACCGCAGCAGGAATAAC

• Codon optimization

The selected organism: Escherichia coli (E. coli) Reason: This is because E. coli is a common model organism for the production of recombinant proteins due to its speed, low cost and ease of manipulation.

• ATGTCTGGTGGAGGTTCTGTTTACCGGCGTTGGTAGTGGTATTCTTGTGGAATTAGATGGCGATGTCAACGGCCACTTTTCCGTTTCAGGCGAGG GTGAGGGAGGCGACTACCGTGTCAAAATCGACACCTTGAAGTTTATTTGCACGACCGGAAAATTGCCTGTACCGTGGACCACACTTTGGTCACTACTG TTATCAACGTGTTCTCGCTATCCGGACCACATGAAGCAGCATGACTTTTAAAAGTGCAATGCCCGAGGGTTATGTTCAAGAGCGGACCATCTTTTTTA AAAGACGACGGCAACTACAAGACGCGCGAGGTGAAGTTCGAGGGCGAACGCGTGGTGAATCGGATTGAGCTAAAAGGAATTGACTTTAAAAGACGACG GCAACATTCTTGGACATAAGCTAGAGTACAATTATAATTCAAACCACGTGTACATCATGGCCGACAAACAAAAAAACGGCATCAAGGTAAACTTTAAA ATTAGACATAATATCGAGGATGGCAGTGTTCAATTAGCCGACCATTACCAACAGAACACCGATAGGCGGACGGTCCTGTATTACCTGACAACCATTAC CTTAGCACGCAGTCTGCACTGTCCAAGGACCCAAATGAGAAACGAGGACCATATGGTGTTGCTAGAGTTCGTTACCGCAGCAGGAATAAC

• Technologies to Produce Protein

Cell-dependent method in this case, focused on E. coli:

Cloning: An insertion of the optimized sequence is carried out in a plasmid.

Transformation: In this step, what is done is that the plasmid is introduced into E. coli.

Expression: With this, an induction is carried out with IPTG to produce GFP.

Purification: This step seeks to use affinity chromatography.

Cell free method: For this purpose, E. coli extracts are used to produce GFP directly.

DNA Read/Write/Edit

DNA Read

(i) What DNA Would You Edit? The DNA to be sequenced would be the F9 gene locus extracted from the patient’s liver cells following CRISPR-Cas9 gene editing. The core purpose is verification — confirming that the correction worked exactly as intended and that no unintended damage was introduced elsewhere in the genome. There are three specific sequencing goals. First, confirming the edit — reading across the exact mutation site to verify that the correct nucleotide was restored by Homology-Directed Repair, and that no indels were introduced by the messier NHEJ pathway instead. Second, off-target analysis — scanning the broader genome for any locations where Cas9 may have made unintended cuts, which is a critical safety check before any edited cells are returned to the patient. Third, monitoring gene expression — by sequencing RNA transcripts (via RNA-seq), it is possible to confirm that the corrected F9 gene is actually being transcribed and translated into functional Factor IX protein, not just sitting silently in the genome. (ii) In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why? Nanopore Sequencing (Oxford Nanopore Technologies) For verifying CRISPR edits in the F9 gene, Nanopore sequencing is the strongest choice. The F9 gene involves complex structural mutations, and Nanopore’s ability to read very long stretches of DNA in a single pass makes it ideal for confirming whether the edit succeeded and detecting any off-target cuts elsewhere in the genome.

1. Generation Nanopore is a third-generation sequencing technology. Unlike first-generation Sanger sequencing (slow, single fragment) or second-generation Illumina (short reads, requires amplification), Nanopore reads single DNA molecules in real time without amplification, and uniquely detects bases electrically rather than optically.
2. Input & Preparation The input is high-molecular-weight genomic DNA extracted from the patient’s edited liver cells. After extraction, DNA quality is checked using NanoDrop and gel electrophoresis. The ends are then repaired and dephosphorylated to create clean blunt ends, followed by ligation of Oxford Nanopore’s proprietary motor protein adapters. Importantly, no PCR amplification is needed, which avoids bias and preserves the DNA’s native state.
3. Sequencing & Base Calling A protein nanopore is embedded in a membrane with a constant ionic current flowing through it. The motor protein feeds the DNA strand through the pore one base at a time, and each base disrupts the current by a characteristic amount. These electrical signals are recorded in real time and translated into a DNA sequence by a neural network-based base calling algorithm such as Dorado.
4. Output The output is a set of long reads — sometimes hundreds of thousands of base pairs — stored in FASTQ format, containing both the sequence and per-base quality scores. These are aligned to the reference genome to confirm the F9 correction, identify any NHEJ-caused indels, and flag potential off-target edits.

DNA Write (i) What DNA Would You Synthesize and Why? The target for synthesis would be a corrected copy of the F9 gene, which encodes Clotting Factor IX — the protein absent or dysfunctional in Hemophilia B patients. Rather than relying solely on CRISPR to repair the mutation in place, synthesizing a complete, healthy F9 sequence allows it to be used as the donor template in Homology-Directed Repair, or delivered independently as a gene therapy construct. The F9 gene spans approximately 34,000 base pairs, and the synthesized version would carry the correct nucleotide at the mutation site, restoring the gene’s ability to produce functional Factor IX. This approach is compelling because it offers a one-time, permanent fix rather than the lifelong Factor IX infusions patients currently depend on.

(ii) What technology or technologies would you use to perform this DNA synthesis and why? Because the F9 gene is too long to synthesize as a single error-free strand using standard chemical methods, the same two-stage approach applies. Silicon-based oligonucleotide synthesis (as used by platforms like Twist Bioscience) prints thousands of short, overlapping DNA fragments in parallel using phosphoramidite chemistry, building the sequence one nucleotide at a time directly onto a chip.

Once detached, these fragments are fed into Gibson Assembly, where an exonuclease exposes overlapping single-stranded ends, a polymerase fills any gaps, and a ligase seals the joins — stitching all the pieces into the complete, corrected F9 sequence in a single reaction. The synthesized F9 construct would then be packaged into an AAV vector (Adeno-Associated Virus), specifically AAV5 or AAV8, which has a strong affinity for liver cells where Factor IX is naturally produced, and delivered intravenously to the patient. The primary limitations are error rates — longer constructs accumulate more synthesis errors, requiring thorough verification with Nanopore sequencing — and the AAV’s limited cargo capacity, which at roughly 4.7kb is smaller than the full F9 gene, necessitating the use of a compact promoter and careful construct design to fit within that constraint.

DNA Edit (i) What DNA would you want to edit and why? The DNA to be edited is the F9 gene located on the X chromosome, which encodes Clotting Factor IX — a protein essential to the blood clotting cascade. Hemophilia B occurs when mutations in this gene prevent the body from producing functional Factor IX, meaning even minor injuries or internal bleeds can become life-threatening without immediate medical intervention. The most common severe mutations include point mutations and small deletions that either truncate the protein or render it completely nonfunctional.

The technology of choice is CRISPR-Cas9, delivered to liver cells via an AAV8 viral vector. CRISPR-Cas9 is the most practical option because it is programmable, relatively affordable, and precise enough to target a single mutation within the F9 gene. AAV8 is paired with it specifically because this serotype has a strong natural affinity for hepatocytes — the liver cells where Factor IX is produced — making intravenous delivery efficient without requiring cells to be removed from the patient. How It Works A custom Guide RNA (gRNA) is designed to match the exact sequence surrounding the F9 mutation. The Cas9 protein escorts this gRNA through the cell, scanning the genome until it finds its complementary sequence and binds. It then executes a Double-Strand Break, cutting through both strands of the DNA helix at the target site. At this point, a donor DNA template carrying the correct F9 sequence is provided alongside the CRISPR machinery. The cell’s repair system uses this template as a blueprint to fix the break through Homology-Directed Repair (HDR), precisely overwriting the mutation with the healthy sequence. Preparation & Input The editing package consists of four components: the Cas9 nuclease, the custom gRNA, the corrective donor DNA template, and the AAV8 vector to carry everything into liver cells. The gRNA must be carefully designed to be unique to the F9 locus to minimize the risk of cutting elsewhere. The entire construct is packaged into AAV8 and delivered intravenously, where it naturally homes to the liver. Limitations The three main limitations are efficiency, off-target effects, and immune response. HDR is inherently inefficient in adult liver cells, as these are largely non-dividing — many cells will default to the imprecise NHEJ repair pathway, potentially worsening the mutation rather than correcting it. Off-target cuts remain a safety concern; if Cas9 mistakes a similar genomic sequence for its intended target, it could disrupt a critical gene. Finally, some patients carry pre-existing immune responses to AAV vectors, which can neutralize the delivery system before it reaches the liver, limiting the therapy’s effectiveness.

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project