Week 1 HW: Principles and Practices

I- 𝙃𝙒 𝙒𝙚𝙚𝙠 1 𝙖𝙨𝙨𝙞𝙜𝙣𝙢𝙚𝙣𝙩 :

1- Application Description:

The biological engineering application I propose is the in silico design of a synthetic metabolic pathway for the biosynthesis of Vitamin B6 in Escherichia coli. The system relies on engineering a non-pathogenic bacterial chassis to overexpress key enzymes of the pdxS/pdxT pathway under tightly controlled genetic regulation.

Vitamin B6 is a water-soluble vitamin from the B vitamin family, it is represented by three main forms: pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM).

It is an essential micronutrient involved in amino acid metabolism, neurotransmitter synthesis, and immune function.

Vitamin B6 is also known as an enzymatic cofactor in its active form, pyridoxal-5′-phosphate (PLP).

Vitamin B6 as a supplement is still largely produced through chemical synthesis, which can be environmentally harmful and resource-intensive. A biologically engineered production system offers a more sustainable, scalable, and environmentally friendly alternative.

Although this project is currently dry-lab only, focusing on computational pathway design, enzyme analysis, and regulatory modeling, it represents a realistic precursor to future wet-lab implementation in academic or industrial contexts.

2- Motivation:

This application is compelling because it:

Addresses a real-world public health and sustainability need.

Integrates molecular biology, synthetic biology, biochemistry, and bioinformatics, areas I am deeply passionate about.

Demonstrates how biological engineering can replace polluting chemical processes.

Raises important governance questions about biosafety, misuse, and environmental responsibility.

3- Governance and Policy Goals for an Ethical Future:

To ensure this application contributes to an ethical future, I focus on the overarching goal of non-malfeasance (preventing harm), alongside constructive and responsible innovation.

High-Level Governance Goals Goal 1: Ensure Biosafety and Biosecurity Prevent accidental or intentional harm arising from engineered biological systems. Sub-goals: Prevent the use of pathogenic organisms or harmful genetic elements Minimize risks of accidental release or misuse Goal 2: Protect the Environment Ensure that biological production systems do not cause ecological harm. Sub-goals: Avoid uncontrolled environmental release Promote sustainable alternatives to chemical synthesis Goal 3: Promote Responsible and Constructive Use Encourage applications that benefit society while maintaining public trust. Sub-goals: Maintain transparency and reproducibility Avoid unnecessary regulatory burdens that impede research 4- Governance Actions: I propose three complementary governance actions, involving different actors and mechanisms.

Option 1: Mandatory Biosafety-by-Design Requirements for Synthetic Metabolic Engineering Purpose Currently, biosafety considerations are often addressed late in the research process. I propose requiring biosafety-by-design principles (e.g., non-pathogenic chassis, inducible systems, genetic containment strategies) at the design stage, even for dry-lab projects intended for future implementation. Design Implemented by: academic institutions, funding agencies Required in: project proposals, course projects, and grant applications Researchers must explicitly justify organism choice, containment strategies, and exclusion of harmful genes Assumptions Researchers will accurately assess risks early Design-stage governance meaningfully reduces downstream risk Risks of Failure & “Success” Failure: becomes a box-checking exercise Success risk: overly conservative designs could discourage innovation

Option 2: Incentivized Transparency and Open Documentation Purpose Promote constructive use by encouraging open-access documentation of synthetic biology designs, models, and assumptions, similar to open-source software or open science initiatives. Design Implemented by: journals, universities, funding bodies Incentives: publication priority, funding advantages, recognition Platforms: GitHub, Benchling, public repositories Assumptions Transparency reduces misuse by enabling peer scrutiny Open science improves reproducibility and trust Risks of Failure & “Success” Failure: sensitive details could be misused Success risk: unequal access may advantage well-resourced actors

Option 3: Tiered Regulatory Oversight Based on Risk Level Purpose Avoid overregulation of low-risk research while maintaining strong oversight of higher-risk applications. Design Implemented by: federal regulators, research institutions Low-risk (dry-lab, non-pathogenic): minimal oversight Higher-risk (wet-lab, environmental release): stricter review Assumptions Risk levels can be accurately classified Institutions have capacity for review Risks of Failure & “Success” Failure: misclassification of risk Success risk: regulatory loopholes exploited by bad actors

5-

6- Prioritized Governance Strategy:

Based on this analysis, I would prioritize a combination of Option 1 and Option 2, supported by Option 3 at the institutional or national level. Option 1 ensures safety and responsibility are embedded early Option 2 promotes openness, trust, and constructive innovation Option 3 provides scalable oversight without stifling low-risk research Trade-offs Considered Balancing transparency with misuse risk Avoiding regulatory overreach that could slow beneficial research Target Audience This recommendation is primarily directed toward: University leadership and course designers (e.g., MIT programs) National research funding agencies International synthetic biology consortia

7- Ethical Reflection:

This week’s material highlighted ethical concerns that were new to me, particularly the idea that design choices themselves are ethical decisions, even before any organism is built. I became more aware of how dry-lab work can still shape real-world outcomes, including environmental and security risks. One key concern is the normalization of increasingly powerful biological tools without proportional attention to governance. To address this, integrating ethics and governance discussions directly into technical training-rather than treating them as an afterthought-appears essential.

8- Some references that helped me:

Wang, et al. Protein engineering and iterative multimodule optimization for vitamin B6 production in E. coli. Nature Communications, 14(5304), 2023

Vitamin B6. Wikipedia (summary of biochemical pathways and commercial synthesis methods).

The pdxS/pdxT genes encode enzymes for the DXP‑independent PLP synthase pathway. This has been studied in multiple bacteria including Streptococcus pneumoniae.

National Academies of Sciences, Engineering, and Medicine (2018). Biodefense in the Age of Synthetic Biology.

Oye, K. A., et al. (2015). Regulation and governance of synthetic biology. Lessons from early years.

9- Note to add:

I note that I used IA (ChatGPT) to enhance the academic writing.

II- 𝗔𝘀𝘀𝗶𝗴𝗻𝗺𝗲𝗻𝘁 (𝗪𝗲𝗲𝗸 𝟮 𝗟𝗲𝗰𝘁𝘂𝗿𝗲 𝗣𝗿𝗲𝗽) :

𝗛𝗼𝗺𝗲𝘄𝗼𝗿𝗸 𝗤𝘂𝗲𝘀𝘁𝗶𝗼𝗻𝘀 𝗳𝗿𝗼𝗺 𝗣𝗿𝗼𝗳𝗲𝘀𝘀𝗼𝗿 𝗝𝗮𝗰𝗼𝗯𝘀𝗼𝗻:

1- DNA polymerase III is the primary replicative polymerase in bacteria known also to be fast and highly processive, responsible for chromosomal DNA replication. Although DNA polymerase I has proofreading activity and can be highly accurate during repair synthesis (high fidelity), it is not the main replicase, Its main function is to remove RNA primers from Okazaki fragments and incorporates nucleotides in some repair pathways (like BER). During the SOS response, specialized polymerases such as DNA polymerase IV and DNA polymerase V perform translesion synthesis and are error-prone, which increases mutation rates. For the DNA polymerase the rate of errors is one error per 10⁵ nucleotides after profreading and MER it can achieve 1 error per 10⁹ to 10¹⁰ nucleotides. The size of humain genome approximately is 3 Gbp (3×10⁹) so 6×10⁹ Nucleotides (Nt). If we consider the rate of error 1 per 10⁹ Nt, the result is 6 mutations per whole genome per replication cycle.

The question is how biology deal with this discrepancy? there are lot of repair pathways first of all the exonuclease activity (3’-5’) of Polymerase that allow an immediately removing of incorrectly incorporated nucleotides. Secondly there are repair pathways that involve many proteins (sometimes a protein become inactivated and no more functional like alkyltransferases proteins) and high energy (e.g: ATP for Helicase…) , I can cite some repair pathways : Base Excision Repair (BER), Nucleotide Excision Repair, Mismatch Repair (MER), Homologous recombination, there is also a system wich is ligh-dependant with the envolve of a specific proteins called photolyases this system is founded in Most bacteria, Archea, some Plants,Fungi and lower eukaryotes, there is also a mutagenic repair pathways called SOS and cell cycle checkpoints to pause replication or trigger apoptosis.

2- the average humain protein lenght is 400 amino acids (aa) A codon is a combination of 3 nucleotides from the 4 that we have so 4³ = 64 codons that code for amino acids. Some amino acids are coded by one codon (3 nt) like Tryptophane,Try,(W) coded by (UGG) and some others amino acids are coded by more than one codon (degeneracy of the genetic code) like Leucine (L) and there is 3 stop points: UGA, UAG, UAA. We assume that our protein is in a lenght of 400 aa so to form it we will have 3⁴⁰⁰ combinations possible of Dna sequences. Not all DNA sequences that encode the same protein work equally well in practice. Cells have a codon usage bias, preferring some codons over others based on tRNA abundance, so using rare codons can slow translation or stall ribosomes. The DNA sequence also determines the mRNA secondary structure, and strong hairpins or loops can reduce translation efficiency. Additionally, some sequences overlap with splice sites or regulatory elements, meaning random codon changes may disrupt proper mRNA processing. Translation speed is important for co-translational protein folding, so using inappropriate codons can produce misfolded or nonfunctional proteins. Finally, certain sequences may create unintended motifs, such as repeats or premature stop codons, further interfering with protein function.

Note That this work is my own and based on my personal understanding and the foundational knowledge I have acquired in molecular biology.

𝙃𝙤𝙢𝙚𝙬𝙤𝙧𝙠 𝙌𝙪𝙚𝙨𝙩𝙞𝙤𝙣𝙨 𝙛𝙧𝙤𝙢 𝘿𝙧. 𝙇𝙚𝙋𝙧𝙤𝙪𝙨𝙩:

1- Current standard: Phosphoramidite solid-phase synthesis DNA nucleotides are added one by one to a growing chain attached to a solid support. Each addition is a chemically controlled reaction, with temporary protecting groups to prevent side reactions. Very efficient for short sequences (oligonucleotides). This is the method used to make standard oligos for PCR, primers, and CRISPR guides.

2- Related to what I Know it’s difficult to make oligo longer than 200 nt beacause of Chemical efficiency limits wich increase with lenght, purification becomes harder as the chain length increases and side reactions and incomplete couplings increase with length.

3- We cannot make it beacause 2000 pb is to high of the practical limit of 200 nt, there will be more errors that will lead to unusables molecules and it’s not reliable. Maybe with PCR and Ligation methods using ligases (I refered to: Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995 Oct 16;164(1):49-53. doi: 10.1016/0378-1119(95)00511-4. PMID: 7590320.)

𝙃𝙤𝙢𝙚𝙬𝙤𝙧𝙠 𝙌𝙪𝙚𝙨𝙩𝙞𝙤𝙣 𝙛𝙧𝙤𝙢 Dr 𝙂𝙚𝙤𝙧𝙜𝙚 𝘾𝙝𝙪𝙧𝙘𝙝:

3- Title: Creation of a Biosensor to Identify Heavy Metals in Environmental Samples in Real Time

The goal of this project is to create a portable, sensitive biosensor that can identify heavy metals like arsenic, cadmium, lead, and mercury in soil and water samples. The biosensor will offer quick, on-site detection with high specificity using genetically modified microbial or cell-free systems in conjunction with fluorescent or electrochemical reporters. The project will evaluate performance in actual environmental samples, describe response dynamics, and optimize sensor design. This work could help public health, regulatory compliance, and environmental safety initiatives by enabling real-time monitoring of heavy metal contamination and offering a scalable and affordable environmental monitoring tool.

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project