Week 1 HW: Principles and Practices

Week # 1 Homework

Principles & Practices

A look at the ethics, safety and security considerations for a biological engineering application with the proposed governance policy goals and actions.

Most countries like Kenya in the developing countries have a waste problem that causes a lot of health issues to the people who live near them while damaging the ecosytem around them that creates a burden for the country in dealing with the financial implications. Synthetic genomic has made it possible through the use of biological organisms that clean up environmental waste and simultaneously produce energy, making this one of the most active fields in biotechnology often referred to as the Circular Bioeconomy. In the latest research which is moving toward Genetically Modified Organisms (GMOs) that can perform multiple tasks at once. Using CRISPR-Cas9, scientists have been able to ceate “super-microbes” that can: • Detect a specific pollutant (like a biosensor). • Break down that pollutant (bioremediation). • Synthesize a fuel molecule (valorization) simultaneously. There is the need to produce biofuels more sustainably than the traditional way with the use of synthetic biology. The problem in Kenya right now we have a lot of second hand clothes that are piled up as waste in dump site, also plastics chocking waterways and scattered all over the streets with no central place to collect them or few collection centers. E-Waste where Kenya generates over 53,000 tonnes annually creating a waste problem. The new technology from synthetic biology would help to eradicate the problem and at the same time generate energy that will help counter the large import bill for gasoline, diesel and kerosine we purchase every year.

The research being done on biological “waste-to-fuel” systems has now led to a major shift from laboratory “proof of concept” to integrated biorefineries where organisms don’t just clean the environment,they act as the living hardware for fuel production. The discovery of a technology through research of Microbial Fuel Cell has made it possible to turn waste into electricity or hydrogen directly without burning anything which is being piloted in waste water plants. Used clothes wastes from Gikomba and Dandora can be turned into Bioethanol,Sewage & Heavy metals from Nairobi River can be turned into Biofuels, Hydrogen or electricity, Plastics in the creation of Bio-oil, Organic waste producing Biomethane. Since the GMO organisms will be bioengineerd to scout for the waste in different damp sites there would be the need to ensure the environment around the site is protected, with the technology being used to make sure the community around benefit from it and have the area restored and once done the organisms can be engineered to sense the completion of there task and intergrate into the ecosystem without harming it.

Environment • How would the damp site be free of the bioengineered organisms after conversion to biofuel? • How will the biofuel be evacuated fron the damp site without harming the ecosystem?

Equitable use of technology • Will the GMO be made available to the public? • How will the technology be used in the area where it is needed and will the community benefit from the biofuel?

Biosecurity • The technology needs to be safe to handle and use without leading to biological disasters. • The GMO should not be able to mutate and create a situation where they alter other organisms in the ecosystem.

Looking at the three different potential governance “actions” with the four aspects below (Purpose, Design, Assumption, Risks of Failure & “Success”)

Researchers • There is the need to show the standards and how the super microbes will be handled and produced either locally or imported. • Publications from reputable institutions to show how they are able to use similar microbes in a safe way with a manual compiled for the laboratory use of them. • A database that has all the known super microbes that are able to produce and how to be handled, the risks and best practices.

Microbiome Companies • There needs to be a way the regulators can look into auditing where the companies are following the law and standards set by researchers. • Public participation is needed to educate the community in the areas where they plan to use their technology. • The need to be informed each stage on what is going on with the project once it commences till the end.

Government Regulators • The agencies tasked to monitor will assess using their standards and gauge on what needs to be done with the super microbes registered on the quantities used. • Always monitoring that safety is observed for the audits conducted abruptly without notice to ensure safety of the products they claim to use and guidelines set. • Ensure the people on the site working are of the recommended number and not overcrowded and following ecological standards and public participation.

The researchers would be the laboratories that test and develop the microbes either in an institution like a University or private entity. The Microbiome companies design the microbiomes and have organisnm engineers who develop new organism using biology,they vary in size from small to large scale. The government regulators look into getting approvals and can use third party firms to enforce the regulations. To get approval in the use of synthetic genomics there are three primary regulators with the process streamlined under the 2022 Genome editing guidelines. The first step is The National Biosafety Authority (NBA) where one gets the permit from for the lab research and the risk assessment. The second step is National Environment Management Authority (NEMA) for the environmental impact assessment and the need for a permit to discharge the treated byproduct and bioprospecting permit for microbes. The third step is The Energy & Petroleum Regulatory Authority(EPRA) where you get the Biofuel production license, Construction Permit and KEBS standardization. The economic risks would be the bioavailability of plastic as an engineered microbe cannot eat a plastic bottle unless it is shredded and pre-treated (Hydrothermal pretreatment). With the introduction of Carbon Credits by the Kenyan governmet in the Climate Change Act,can lead to a saving of 30% towards the operational costs.
Based on the scoring above the goverment would need to know how the super microbes function and have the community know the benefits of the use of them in clearing the waste. The Researchers and Microbiome companies need to return at least 5-10 % of the biofuels as a way for giving back to the community so as to minimize pushback. Since there is the incentive offered by the government on the use of local microbe,research can be done to see how they can be engineered to reduce the initial set up cost.

Homework Answers for Professor Jacobson

1. Nature’s machinery for copying DNA is called polymerase.

What is the error rate of polymerase? The error rate of polymerase is 1 mistake per 10*6 base pairs.

How does this compare to the length of the human genome? DNA polymerase which is an enzyme is approximately 10 – 15 nanometers (nm) in length while the human genome which is the template is approximately 2 meters when stretched out. A scale ratio of 1: 108 x longer (200m /10 nm = 20,000,000x)

How does biology deal with that discrepancy? It does this by not relying on a single enzyme. It uses a highly organized, factory-like system with four key strategies: The first is the multiple origins of replication instead of a single one, each origin has two replication forks creating a replication bubble where thousands of DNA polymerase molecules can work simultaneously across all chromosomes where each has a small copying segment.

The second, is it doesn’t work alone as is part of a complex of proteins known as a replisome a key component is the sliding clamp (PCNA in humans) where the donut- shaped protein encircles the DNA and tethers the polymerase to the template which leads to the increase in processivity making one polymerase can be able to add thousands of nucleotides without falling off, turning it from a slow inefficient enzyme to a high speed, long distance replicator.

Third, different polymerases have specialized roles with the leading strand being synthesized continuously by a highly processive polymerase and the lagging strand synthesized in short Okazaki fragments that require different coordinated processes. The main replicative polymerases have proofreading ability (3’→5’ exonuclease activity). Where they are able to mmediately back up and fix a mistake, ensuring speed doesn’t come at the cost of catastrophic error rates (final error rate: ~1 in 10 billion bases)

Fourth, the compartmentalization and packaging of DNA has mabe the 2 meters of DNA not in a loose tangle. It istightly wound and packaged with proteins into chromosomes inside the nucleus (~10 µm wide) where the replisome has to navigate this dense chromatin structure, with the Helicases unwinding it, topoisomerases relieve twisting stress, and other proteins modify the packaging to allow access. This organization makes it possible to bring distant genomic regions into physical proximity while making the logistics of finding origins and assembling machinery more efficient.

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 don’t work to code for the protein of interest? The different ways to code an average human protein is 10*214 , which is a very large number and impractical. Most of the codes fail in producing a functional protein in the living cell. The reason why most of the codes don’t work is not all of them are created equal where codon usage bias can affect translaton speed leading to misfolded or incomplete proteins. The second, is where mRNA has a secondary structure where it folds back on itself and form shapes like hair pins and loops and if a randomly chosen DNA sequence creates an mRNA strand that folds at the beginning, the ribosome can’t get on the track and start reading. The third is splicing (cutting and pasting) RNA before it is translated where specific sequences that signal where an intron (junk DNA) finishes ans an exon(coding DNA) starts, with many containing the code for a “splice-site” in the middle of the gene and throw away, leaving a fragment which is a useless protein. The fourth, DNA is chemically stable cause of the G-C pairs, where it is held together by hydrogen bonds, G-C pairs have three bonds, while A-T pairs only have two. Where too high of a G-C content the DNA ‘zipper’ would be really hard to open whereas a low A-T rich would make it unstable. The sixth is the human immune system through evolution, recognizes the Cytosine followed by a Guanine (CpG) as a pattern of a viral or bacterial infection and if the code has too many of these “CpG islands” triggers the cell that its under attack by a virus and could lead to gene silencing or an inflammatory response to “shield” itself.

Homework Answers for Dr. LeProust

1. What’s the most commonly used method for oligo synthesis currently? The oligonucleotide synthesis is the phosphoramidite method, using thr Solid Phase Synthesis (SPS). Naturally DNA builds in the 5’ to 3’ direction, while laboratory method builds the chain backward—from the 3’ end to the 5’ end. The method has a four step cycle, where it happens on a solid support (usually controlled-pore glass or polystyrene beads). The addition of a single nucleotide, the machine must complete a full revolution of these four chemical steps:

The first step known as Deblocking (Deprotection) where the nucleotide is already attached to the solid support. Its 5’-hydroxyl group is “blocked” by a protective chemical called DMT (dimethoxytrityl) to prevent it from reacting prematurely. An acid is added to wash away the DMT, leaving a “naked” 5’-OH group ready for the next link. The second step whrere the next nucleotide (a phosphoramidite monomer) is added to the chamber along with an activator. The 5’ end of the growing chain binds to the 3’ end of the new monomer. Leading to an Efficiency usually >99%, but in chemistry, 100% is impossible. The third step is capping where a small percentage of the chains that failed to couple in Step 2, they must be “capped” (usually with acetic anhydride). This prevents them from reacting in future cycles, which would result in “deletion mutants”—strands that are missing a middle letter. The fourth step is oxidation where a bond formed during coupling is a bit unstable (a phosphite triester). An iodine solution is added to oxidize this bond, turning it into a stable phosphate triester which is the familiar backbone of DNA.

2. Why is it difficult to make oligos longer than 200nt via direct synthesis? The phosphoramidite synthesis has a specific limit where errors accumulate with every step, making it hard to build a single strand longer than 200–300 bases with high purity. Since every time you add a base, there is the of lose a tiny bit of your starting material because the reaction never goes to 100% completion.

Secondly, the Capping step of the cycle stops “failed” chains from growing further which leads to Purifying the “perfect” sequence away from the “almost perfect” ones becoming a nightmare. Comparing it to trying to find a specific grain of sand in a pile of slightly smaller grains of sand Thirdly, The first step of the cycle (Deblocking) uses an acid to remove the protective DMT group that DNA doesn’t actually like. Every time you expose the growing chain to acid, there is the risk of depurination where you accidentally snip a Guanine or Adenine base off the sugar backbone. If for comparison, a 200nt strand being subjected to the first base to 200 rounds of acid wash. By the time you reach the end, the beginning of your sequence is often chemically “chewed up.” Fourth, the time and mechanical failure as synthesizing 200 bases takes a long time (often 10–15 hours) and the longer the run, the higher the possibility of a “mechanical failure” where a bubble in the line, a slight drop in temperature, or a reagent running dry. Bringing about a failure at base 190 leading to wasting the entire 14-hour run and all the expensive chemicals used up to that point.

3. Why can’t you make a 2000bp gene via direct oligo synthesis? First, its statistically impossible even if your machine was the most efficient running 99.5% efficiency per base addition where only 0.004% of the molecules in your final mixture would actually be the correct 2,000bp length. The other 99.996% would be “truncated” sequence with broken fragments that are missing one or more bases.The massive mound of chemical errors makes it impossible to find the perfect bases.

Second, there is the physical crowding and stuttering while the DNA chain grows to 2,000 bases, it doesn’t just stay a neat and organized. It starts to fold, tangle, and stick to the solid support (the glass or plastic bead it’s being built on). This leads to the “top” of the growing DNA chain becomes physically hard for new chemicals to reach because it’s buried in a crowd of other DNA strands. At the addition of the 2,000th base, the 1st base has been washed in acid 2,000 times making the chemical integrity of the beginning of the gene completely compromised.

Homework Answers for George Church

1. (Using Google & Prof . Church’s slide #4) What are the 10 essential amino acids in all animals and how does this affect my view of the “ Lysine Contigency”? Animals require 10 essential amino acids from their diet since they cannot synthesize them. These are universally needed across all animals like mammals, birds, and fish for protein synthesis and growth.

Essential Amino Acids List The 10 essential ones, remembered by the acronym “PVT TIM HALL,” are:

• Phenylalanine
• Valine
• Tryptophan
• Threonine
• Isoleucine
• Methionine
• Histidine
• Arginine
• Leucine
• Lysine

Lysine Contingency as explained in “Jurassic Park”, was the “lysine contingency” genetically modified dinosaurs to be unable to produce lysine, an essential amino acid, making them dependent on park-supplied supplements to prevent escape and survival in the wild. It failed scientifically as all animals, including dinosaurs as modeled, already couldn’t synthesize lysine and obtain it from protein-rich foods like meat or plants, abundant in ecosystems. Removing synthesis offers no control, as lysine is widespread, rendering the plan ineffective as dinosaurs would simply eat lysine-containing prey or vegetation.

References

Bioremediation of environmental wastes (https://www.frontiersin.org/journals/agronomy/articles/10.3389/fagro.2023.1183691/full) Community Guide to Bioremediation (https://semspub.epa.gov/work/HQ/401583.pdf) Global Situation of Bioremediation of Leachate-Contaminated (https://pmc.ncbi.nlm.nih.gov/articles/PMC10145224/) (https://pmc.ncbi.nlm.nih.gov/articles/PMC11607652/) Conversion of organic wastes into biofuel by microorganisms ( https://www.sciencedirect.com/science/article/pii/S2772801323000180) (https://envaco.org/epr-in-kenya-legal-framework-and-policy-instruments/) (https://envaco.org/e-waste-management-in-kenya-challenges-and-opportunities-in-the-digital-age/) (https://nutrenaworld.com/blog/horses/what-are-essential-amino-acids-in-protein-and-why-do-they-matter/) (https://jurassicpark.fandom.com/wiki/Lysine_contingency)

Week 2 DNA READ, WRITE, AND EDIT

Week # 2 Homework

DNA READ, WRITE & EDIT

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project