Week 1 HW: Principles and Practices

Application Idea:

The development of an engineered bacterial biosensor for real-time hydration detection as a preventive health measure in aging populations.

An engineered skin bacterium, applied as a lotion on the wrist or forearm, could detect body hydration levels and generate an electric current detectable by an electronic wearable component.

Why develop this application?

Water is vital for health, and yet, neglecting hydration is common. The elderly are particularly vulnerable. Dehydration perturbs the gastrointestinal (GI) tract, leading to difficulty passing stool and overall adverse effects on GI health.

The idea for this project was inspired by a non-invasive detection method that works via contact with skin that can be sampled for several parameters, not exclusively for hydration. Others, such as amino acids for nutritional status and glucose for insulin levels, can be measured and are directly read out from the body’s blood.

The skin readout for the body’s hydration status, as well as mental activity, has recently been developed by the University of California, Berkeley researchers (Kim et al 2025). The method is based on a microfluidic sensor device that uses the skin’s electrical property, the electrodermal activity.

The proposed project uses the synthetic biology approach, a biosensor that can report the body’s hydration status in real-time. Technologies for building such a biosensor are out there, such as the utility of the osmolarity-responsive operon (Rashid et al 2023) and the electric current generator, which is a synthetic electron transport chain (Atkinson et al 2022), but engineered skin bacteria as biosensors have not been made specifically for health preventive measures. This project is aimed at testing the bacterial commensal organisms as biosensors working via the skin.

References:

Kim, S-R., Y. Zhan, N. Davis, S. Bellamkonda, L. Gillan, E. Hakola, J. Hiltunen, and A. Javey. 2025. Nature Electronics.

Rashid, F-Z. M., F. G. E. Cremazy, A. Hofmann, D. Forrest, D. C. Grainger, D. W. Heermann, and R. T. Dame. 2023. Nature Communications.

Atkinson, J. T., L. Su, X. Zhang, G. N. Bennett, J. J. Silberg, and C. M. Ajo-Franklin. 2022. Real-time bioelectronic sensing of environmental contaminants. Nature.

Major governance policies

1. Establishing genome repositories for synthetic microbiome species
2. Ensuring do not release through biocontainment strategies
3. Adopting validation studies as an alternative to animal testing
4. Comply with regulatory on human subjects’ clinical trials
5. Documenting product safety through environmental toxicology studies
6. Implementing incentives and educational workshops

Establishing genome sequence repositories for synthetic microbiome species

Bacterial species used in this project, isolated and sequenced from the skin microbiome, should be recorded according to the general rules applies to biological agents. Any variations made into the organism through recombinant DNA techniques, including the introduction of DNA from other sources, should be recorded to comply with biosecurity rules.

Ensuring do not release through biocontainment strategies

Genetically modified organisms should not be released to the environment. Biocontainment strategies should be in place to ensure that the genetically engineered organism, for therapeutic interventions, cannot survive in the environment. One way to do this is through codon engineering, specifically for a non-canonical amino acid, which would create a dependency for the unnatural amino acid, which is lacking in the environment.

Subgoal: Non-canonical amino acids are expensive. Biomanufacturing cost will increase due to the need for that substrate. One way to reduce the cost is to have on-site manufacturing of non-canonical amino acids from precursors. Because precursors can be toxic, the manufacturing of chemicals needs to comply with local regulatory rules, such as building and equipment and engineering requirements.

Adopting validation studies as an alternative to animal testing

As an alternative to animal testing, validation studies should be based on artificial organoid-based systems. This ensures cruelty-free ethical conduct. Artificial skin models are already being developed and are available for monitoring the interstitial fluid compartment. Artificial 3D print skin models with built-in complexities, such as immune cells, could provide a setup for an initial understanding of the performance of the biosensor.

Comply with regulatory on volunteering human subjects’ clinical trials

Live organisms cannot be tested on humans without following laws, regulations, and guidelines applicable at the national and international levels. To demonstrate the clinical efficacy of the live organism, proper documentation and requesting permission should be established for the approval process.

Implementing incentives and educational workshops

General public acceptance of genetically modified organisms (GMOs) applied to the skin may be received with resistance. Educational workshops and materials should be available to the general public, as they introduce commensal microorganisms on skin and their genetic manipulation for biocontainment. A way to encourage participation is to implement an incentive system, such as a subscription with health insurance that includes paid benefits.

Based on the above, I would prioritize establishing regulatory policies regarding:

1. Generating genome sequence repositories,
2. Ensuring do not release through biocontainment applications,
3. Documenting product safety through environmental toxicology studies

Although it is not a priority, general public education on the use of engineered organisms to monitor biomarkers is important because it would create buy-in for health preventive products.

Assignment Week 2 Lecture Prep

Homework questions from Professor Jacobson:

1. Nature’s 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?

Depending on the polymerase, the error rate ranges from 10⁻⁶ to 10⁻⁴.

The human genome length is 3.2 x 10^9 bp.

We could expect at least 3.2 × 10^3 errors occurring per replication.

There are multiple mechanisms employed to deal with this discrepancy:

• a. base selection
• b. proofreading
• c. mismatch repair

Mutation rates are lowest when 3 mechanisms are working together. It is reported that the mutation rate per base pair in prokaryotes is 2.6 x 10⁻¹⁰ and in eucaryotes is 3.3 x 10⁻¹⁰.

• The initial base selection ensures incorporation of the correct nucleotide 200,000 to 2000,000 times more likely.
• Having a proofreading activity, the polymerase can detect errors and uses exonuclease activity to remove incorrect nucleotides, improving fidelity by 40 to 200-fold.
• The mismatch repair system detects mismatches that escaped proofreading and catalyzes excision and synthesis, improving fidelity by 20 to 400-fold.

References:

R.M. Schaaper. 1993. Base selection, proofreading, and mismatch repair during DNA replication in Escherichia coli. The Journal of Biological Chemistry.

I’ve used lecture slides and Claude’s search.

2. How many different ways are there to code DNA (DNA nucleotide code) for an average human protein? In practice, what are some of the reasons that all of these different codons don’t work to code for the protein of interest? There are 64 codon combinations to code for all 20 amino acids. Most amino acids have more than 1 code, except methionine and tryptophan. Leucine, serine, and arginine have 6 different codons each.

An average human protein of about 469 amino acids can be coded by many combinations of DNA sequences that are too many to count. But not all coding would work. Some of the reasons are the following:

In practice, synonymous codons won’t work because of codon bias, which is organism-specific. Also, synonymous codons’ corresponding tRNAs might be less abundant in a given organism. In this situation, rare codons will have slow translation efficiency. Protein folding efficiency would also be slow. Synonymous codons may also cause mRNA instability.

References:

I’ve used lecture slides and Claude’s search.

Homework questions from Dr. LeProust:

1. What is the most commonly used method for oligo synthesis currently?

The most commonly used method for oligo synthesis currently is the phosphoramidite method developed in the 1980s.

2. Why is it difficult to make oligos longer than 200 nt via direct synthesis?

It is difficult to make oligos longer than 200 nt via direct synthesis because of the accumulation of errors, leading to a greater percentage of the product being truncated.

3. Why can’t you make a 2000 bp gene via direct oligo synthesis?

A 2000 bp gene cannot be made via direct oligo synthesis due to the accumulation of errors. A direct synthesis is limited to 200 nt. A 2000 bp gene can be made through the enzymatic assembly of shorter pieces by PCR.

References:

I’ve used lecture slides and Claude’s search.

Homework questions from Professor George Church:

Using Google & Prof. Church’s slide #4:

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

The following amino acids are essential in all animals: His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, and Arg.

Protein synthesis by the animal is more sensitive to lysine deficiency because animal proteins are rich in lysine; a slight decrease in lysine intake can influence the rate of protein synthesis (Ball et al 2007). I learned that not all proteins are rich in lysine; proteins in plants and grains are low in lysine (Mathews 2020), unlike proteins from animal sources.

As I read about the following study conducted in Pakistan, I believed in the importance of lysine supplementation in the human diet. A study conducted in Pakistan showed that wheat flour fortified with lysine improved the weight and height of children as compared to a control group (Hussain et al. 2004).

References:

Ball, R. O., K. L. Urschel, and P. B. Pencharz. 2007. Nutritional consequences of interspecies differences in arginine and lysine metabolism. The Journal of Nutrition.

Mathews, D. E. 2020. Review of lysine metabolism with a focus on humans. The Journal of Nutrition.

Hussain, T., S. A. Mushtaq, A. Khan, and N. S. Scrimshaw. 2004. Lysine fortification of wheat flour improves selected indices of the nutritional status of predominantly cereal-eating families in Pakistan. Food and Nutrition Bulletin, The United Nations University.

I’ve used lecture slides, ChatGPT, and Google Scholar search.

Prompts:

“What are the essential amino acids in all animals?”

“ Can you research the lysine contingency?”