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 4 HW: Protein Design Part 1

Conceptual Questions

— Q1: How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons)

First, we need to calculate the number of moles and multiply by Avogadro’s number (NA=6.022×1023 mol−1). An amino acid has an average mass of ~100 Daltons (Da), which is roughly equivalent to 100 g/mol. Meat is mostly protein (~20% of its weight is protein). → 500 g of meat contains approximately 100 g of protein. Since 1 mole of amino acids weighs ~100 g, there are ~1 mole of amino acids in 100 g of protein. → 1 mole is equivalent to 6.022 × 10^{23 molecules (Avogadro’s number). So you consume approximately 6 × 10}23 amino acids in 500 g of meat.

Q2: Why do humans eat beef but do not become a cow, eat fish but do not become fish?

When you consume animal proteins — whether from beef, fish, or any other source — your body does not simply absorb and repurpose those proteins wholesale. Instead, the digestive system dismantles them through a carefully orchestrated process involving enzymes such as pepsin and various proteases, which hydrolyze the peptide bonds holding amino acids together, reducing complex foreign proteins into their simplest building blocks: individual amino acids and small peptides. These generic molecular units are then absorbed through the intestinal lining into the bloodstream, where they are transported to cells throughout the body. Once inside the cell, the process shifts from digestion to construction, guided by the Central Dogma of molecular biology — the principle that genetic information flows from DNA to RNA to protein. Using the instructions encoded in human DNA, the cell’s ribosomes read messenger RNA transcripts and reassemble those same basic amino acids into entirely new, distinctly human proteins. This elegant process explains why eating cow or fish protein does not make you more “cow-like” or “fish-like” — because by the time those amino acids are rebuilt into proteins, they are following your body’s own genetic blueprint, not the animal’s.

Q3: Why are there only 20 natural amino acids?

The 20 protein amino acids were chosen during evolution for their chemical stability, functionality and compatibility with the ribosomal machinery. Furthermore, their structure allows them to perform a wide variety of enzymatic and structural functions without being excessively complex.

Q5: Where did amino acids come from before enzymes that make them, and before life started?

Long before life existed on Earth and biological enzymes evolved to synthesize proteins, amino acids were being formed through entirely abiotic — or non-living — chemical processes. The landmark Miller-Urey experiment provided the first compelling laboratory evidence for this, demonstrating that when the conditions of early Earth are simulated — a reducing atmosphere composed of methane, ammonia, hydrogen, and water vapor — and electrical discharges are introduced to mimic lightning strikes, amino acids spontaneously emerge from inorganic chemistry alone. This groundbreaking result suggested that the fundamental building blocks of life were not the product of biology, but rather an inevitable outcome of basic chemistry under the right environmental conditions. Further supporting this idea, astrobiological research has revealed that amino acids are not exclusive to Earth — they have been discovered on carbonaceous chondrite meteorites, most notably the Murchison meteorite, which was found to contain a diverse array of extraterrestrial amino acids.

Q6: If you make an α-helix using D-amino acids, what handedness (right or left) would you expect?

If a protein were constructed entirely from D-amino acids rather than the L-amino acids found in nature, you would expect it to fold into a left-handed α-helix. In natural proteins, L-amino acids preferentially adopt right-handed α-helical conformations because this geometry minimizes steric clashes between the side chains and the peptide backbone, making it the energetically favorable configuration. D-amino acids, however, are the exact mirror images — or enantiomers — of their L counterparts, meaning their stereochemical constraints are precisely reversed. As a result, a polymer built entirely from D-amino acids would experience the same stabilizing forces and steric considerations as a natural protein, but reflected in the opposite direction, driving the chain to fold into a left-handed helix instead. This mirror-image relationship illustrates just how profoundly the chirality of individual amino acids influences the three-dimensional architecture of the proteins they compose, and highlights why the near-universal selection of L-amino acids in biological systems is so fundamental to the structural consistency of life as we know it.

Q7 Can you discover additional helices in proteins?

Beyond the well-known α-helix, proteins and engineered peptides can adopt a surprising variety of helical conformations, and scientists have developed several creative strategies to discover and design them. While naturally occurring proteins predominantly utilize the standard α-helix, the 3-helix, and the rarer π-helix, the conformational space available to polypeptide chains is far broader than what evolution has sampled. One powerful approach involves the use of unnatural amino acids — such as β- or γ-amino acids, which carry extra carbon atoms in their backbone — to build synthetic peptides called foldamers that fold into entirely novel helical geometries, such as the 14-helix or 12-helix, structures with no natural counterpart. Another strategy exploits stereochemistry by alternating D- and L-amino acids within a single chain, producing unusual structures like the hollow tubular helix found in the antibiotic Gramicidin A, which behaves fundamentally differently from the solid cylinder of a conventional α-helix. Finally, computational de novo design tools such as Rosetta allow structural biologists to mathematically explore novel hydrogen-bonding networks and backbone torsion angles, effectively engineering stable helical architectures that nature never arrived at through evolution. Together, these approaches reveal that the helices found in natural proteins represent only a small fraction of what is geometrically and chemically possible.

Q8 Why are most molecular helices right-handed?

Most molecular α-helices are right-handed because biological systems exclusively use L-amino acids. In a right-handed helix made of L-amino acids, the side chains (R-groups) point outward and downward, which minimizes steric hindrance (spatial crowding) with the carbonyl oxygen atoms of the polypeptide backbone. If L-amino acids were forced into a left-handed α-helix, the side chains would structurally clash with the backbone, creating a high-energy, thermodynamically unstable state.

Q9 Why do β-sheets tend to aggregate? What is the driving force?

The tendency of β-sheets to aggregate is driven by two primary forces that act in concert to pull individual sheets together into larger, often insoluble structures. The first is the presence of unsatisfied hydrogen bonds along the exposed edges of each β-sheet, where unpaired amide donors and carbonyl acceptors remain highly reactive and eagerly seek out complementary partners on neighboring sheets, effectively stitching them together through intermolecular hydrogen bonding. The second driving force is the hydrophobic effect, which arises from the amphipathic nature of many β-sheets — one face is hydrophilic while the opposing face is hydrophobic. In an aqueous environment, the hydrophobic faces are thermodynamically compelled to shield themselves from surrounding water molecules, driving them to pack tightly against one another and stack into larger fibrillar assemblies. Together, these two forces create a powerful thermodynamic pull toward aggregation, which has significant biological consequences — this very mechanism underlies the formation of amyloid fibrils, the insoluble protein aggregates associated with devastating neurodegenerative diseases such as Alzheimer’s and Parkinson’s, where normally soluble proteins misfold and their exposed β-sheet edges and hydrophobic faces drive runaway aggregation in the cell.

Q10 Why do many amyloid diseases form β-sheets? Can you use amyloid β-sheets as materials?

The prevalence of β-sheet misfolding in diseases like Alzheimer’s is rooted in the extraordinary thermodynamic stability of a structure known as the cross-β spine, in which tightly interdigitated β-strands form a rigid, highly ordered fibril that represents a deep low-energy state. Once a protein misfolds into this conformation, it does not simply remain an isolated aberration — it acts as a template, recruiting and converting neighboring normal proteins into the same misfolded state in a self-propagating chain reaction that progressively builds insoluble amyloid plaques. This prion-like mechanism makes amyloid formation particularly insidious, as the thermodynamic favorability of the fibril structure means the process is essentially irreversible under physiological conditions. However, the same properties that make amyloid fibrils so destructive in disease also make them remarkably attractive as engineering materials. Their mechanical strength is exceptional, rivaling that of spider silk and steel at the nanoscale, and their resistance to biological degradation gives them a durability that few natural materials can match. Recognizing this potential, engineers and materials scientists have begun harnessing modified, non-toxic amyloid sequences to design a wide range of advanced materials, including nanomaterials, hydrogels, biosensors, and scaffolds for tissue engineering — effectively repurposing one of biology’s most feared structural motifs into a platform for cutting-edge biotechnology.

Week 6 HW: GENETIC CIRCUITS PART I: ASSEMBLY TECHNOLOGIES

1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

The Phusion Master Mix is basically a ready-to-use mix that makes setting up PCR way easier since everything is already in it. The main component is the Phusion High-Fidelity DNA Polymerase, which is the enzyme that actually copies the DNA. What makes it special is that it catches and fixes mistakes as it goes, giving you really accurate amplification. It also has the four dNTPs which are the building blocks the polymerase uses to build new DNA strands. There’s also a reaction buffer that keeps the pH and salt conditions stable so the enzyme works properly.

2.What are some factors that determine primer annealing temperature during PCR?

The annealing temperature matters a lot because if it’s too low, your primers bind nonspecifically and you get messy results, and if it’s too high, they won’t bind at all. The most important factor is the melting temperature (Tm) of your primers — the annealing temperature is usually set about 3–5°C below the lower Tm of the two primers. Primer length plays into this too since longer primers have higher Tm values. GC content is another big one — G-C pairs have three hydrogen bonds instead of two, so GC-rich primers are more stable and need higher temperatures to melt.

3.There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other.

Both methods can give you linear DNA fragments, but they work pretty differently. PCR uses primers, a polymerase, and thermal cycling to amplify a specific region. The big advantage is flexibility — you can design primers to add any sequence you want to the ends of your fragment, like overlaps for Gibson assembly. The downside is there’s some risk of mutations, though high-fidelity polymerases like Phusion make this pretty minimal. Restriction digestion, on the other hand, cuts DNA at specific recognition sequences using enzymes, and it’s done at a constant temperature.

4.How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

Gibson assembly works by joining fragments that share overlapping sequences at their ends (usually around 20–40 bp), so you have to make sure those overlaps are designed correctly. For PCR fragments, you add the overlap sequences directly into the 5’ tails of your primers — so the forward primer of one fragment carries the end of the previous fragment, and so on. For restriction-digested fragments, you need to check that the ends left by the enzyme line up with the overlap region of the adjacent fragment. The best way to check everything is to model the assembly in software like Benchling before you even start — you can simulate how all the fragments will come together and catch any design errors early. Basically, if the end of fragment A perfectly matches the beginning of fragment B, you’re good.

5.How does the plasmid DNA enter the E. coli cells during transformation?

E. coli cells don’t naturally take up DNA, so you have to make them “competent” first. With chemically competent cells, they’re treated with CaCl₂, which neutralizes the negative charges on both the DNA and the cell membrane. Then you do a heat shock which temporarily disrupts the membrane and lets the plasmid get in. With electrocompetent cells, it’s a bit different: a brief electroporation creates tiny pores in the membrane that the DNA can pass through. Both methods are essentially creating a temporary opening in the membrane for the DNA to enter.

6.Describe another assembly method in detail (such as Golden Gate Assembly) Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).

Golden Gate Assembly is a really elegant method that lets you assemble multiple DNA fragments in a single reaction. It uses Type IIS restriction enzymes — like BsaI — which are special because they cut outside of their recognition sequence. So you can engineer your fragments so that after the enzyme cuts, it leaves behind a specific 4-bp overhang that you designed, and the recognition site itself gets removed. When you run the digestion and ligation at the same time in one tube, the correctly assembled fragments stick together through their matching overhangs and get ligated. Any fragments that aren’t assembled correctly still have the recognition site, so they just get cut again.

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

Heavy Metal Detection and Removal Using Biosensor Microbes

🔬 Project Documentation

SECTION 1: ABSTRACT

Significance: Heavy metal contamination, specifically mercury ($Hg^{2+}$), poses a critical threat due to its persistence and bioaccumulation in living organisms. In industrial and mining regions like Lima, Peru, traditional detection relies on expensive analytical equipment (ICP-MS), which is inaccessible to local communities. This project addresses the urgent need for a low-cost “sense-and-respond” biological system.

Broad Objective: The overall goal of this project is to engineer Pseudomonas putida to serve as a dual-action biosensor and bioremediator that can simultaneously detect mercury concentrations and initiate physical sequestration.

Hypothesis: This project tests the principle that a MerR-regulated genetic circuit can exhibit high sensitivity to $Hg^{2+}$ ions, triggering a proportional expression of Green Fluorescent Protein (GFP) for quantification and SmtA (a bacterial metallothionein) for sequestration.

Specific Aims:

1. Characterize the sensitivity of the MerR-based sensing module.
2. Engineer a surface-display module for metal binding.
3. Develop a strategy for community-led deployment in contaminated sites.

Methods: The technical approach utilizes Golden Gate Assembly to create a bicistronic operon under the control of the mercury-responsive $P_{mer}$ promoter. Detection is measured via fluorometry (488nm/509nm). Remediation is achieved through the expression of cysteine-rich Metallothioneins (MTs). These proteins use thiol-group coordination to bind $Hg^{2+}$ ions with high affinity. To prevent cellular toxicity, these proteins are fused to the Lpp-OmpA system for display on the outer membrane of the cell.

SECTION 2: PROJECT AIMS

Aim 1: Experimental Aim (The Sensing Circuit)

The first aim of my final project is to construct and validate a mercury-responsive genetic circuit in Pseudomonas putida by utilizing the MerR regulatory protein and the $P_{mer}$ promoter. MerR acts as a specialized transcription factor; when $Hg^{2+}$ binds to its conserved cysteine residues, it induces a conformational twist in the DNA, allowing RNA polymerase to initiate transcription of the GFP reporter. This aim will establish the operational detection limit of the biosensor.

Aim 2: Development Aim (The Binding Module)

The next step following the successful creation of the sensor is to engineer a remediation module by expressing SmtA Metallothioneins. These proteins are highly enriched in Cysteine (Cys) residues, which provide sulfur atoms for stable coordination of heavy metals. To maximize capture efficiency, I will utilize a surface-display strategy (fusing SmtA to the OmpA protein) so the bacteria can sequester mercury directly from the external environment without requiring intracellular transport.

Aim 3: Visionary Aim (Autonomous Remediation)

The long-term vision is to democratize environmental monitoring by deploying these microbes in a “Living Filter” system. This involves immobilizing the engineered P. putida in a porous matrix to create a self-regenerating water treatment device. This challenges the current paradigm of centralized waste management by providing local communities with a cost-effective, autonomous method for both monitoring and cleaning their water sources.

SECTION 3: BACKGROUND

Background and Literature Context

The MerR protein is a highly specific metalloregulatory protein. Research by Brown et al. (2003) demonstrates that MerR binds to the $P_{mer}$ operator as a homodimer; upon mercury binding, it rotates the DNA to activate transcription. For sequestration, Metallothioneins (MTs) like SmtA are utilized. As discussed by Romero-Isart and Vasak (2002), these proteins form metal-thiolate clusters, allowing a single protein to bind multiple mercury ions with high stability. Currently, most technologies offer “sensing only” or “remediation only”; an integrated, field-robust system in a resilient chassis like P. putida is a notable gap in current research.

Innovation and Novelty

This project is innovative because it integrates detection and remediation into a single, automated “sense-and-respond” circuit.

• Integrated Logic: Using a bicistronic design ($P_{mer} \to GFP + MT$) ensures that remediation effort is strictly proportional to the contamination detected.
• Surface Display: Instead of internalizing toxins, the project uses Lpp-OmpA fusions to keep mercury on the cell exterior, protecting the host’s metabolism and making metal recovery easier.
• Host Selection: By utilizing P. putida instead of E. coli, the project applies synthetic biology to a “non-model” organism that is naturally optimized for survival in harsh industrial waste.

Impact and Significance

• The Problem: Mercury is a “forever toxin” that accumulates in the food chain, causing neurological damage in local populations.
• Societal Contribution: This project provides a “Green Chemistry” alternative to chemical precipitation, creating a low-cost tool for environmental justice in marginalized mining communities.
• Knowledge Advancement: Achieving these aims will provide critical data on the metabolic cost of maintaining dual-action circuits in environmentally-robust bacteria.
• Field-level Change: If successful, this changes the field from passive monitoring to active surveillance, where cleanup and detection occur simultaneously and autonomously at the source of contamination.

🧬 System Architecture

[ merR Regulator ] --(Constitutive)--> [ MerR Protein ]
                                            |
                                            v
[ Pmer Promoter ] --(Induced by Hg2+)--> [ RBS ] --> [ GFP Reporter ] --> [ RBS ] --> [ SmtA-OmpA Fusion ]

Group Final Project