Week 1 HW: Principles and Practices

1. Bioengineering Application / Product to Be Developed

Bioengineering Product

A bioplastic derived from organic waste that is composed of 100% biopolymers, food-grade, and naturally biodegradable. This product is designed as an alternative to fossil-based polymer plastics that are widely used today, particularly for single-use packaging applications.

Key Product Specifications

• Main raw materials Sourced from underutilized organic waste, such as:

o cassava peels

o sugarcane bagasse

o coconut coir

o other organic biomass rich in starch, cellulose, or lignin

• Material composition

o The matrix, plasticizer, and filler are entirely derived from natural biopolymers

o No fossil-based synthetic additives are used

• Product characteristics

o Mechanical and physical properties comparable to conventional plastics

o Safe for human health and compliant with food-grade standards

o Fully bio-based

o Easily biodegradable without leaving harmful residues

Rationale for Product Development

• Single-use plastic remains one of the most significant environmental challenges, and effective solutions to reduce its use are still limited.

• Indonesia has abundant organic waste and rich biodiversity that have not been optimally utilized as sustainable raw materials.

• The development of this bioplastic is expected to:

o reduce dependence on fossil-based plastics

o increase the value of organic waste

o promote a circular economy based on local resources.

2. Governance Goals

Primary Governance Goal

To ensure that this bioplastic serves as a viable alternative to fossil-based polymer plastics without creating biological, social, or environmental risks, while supporting an ethical and sustainable future.

Sub-Goals

1) Ensuring the Principle of Non-Maleficence

• Ensuring that all bioplastic components:

o are non-toxic

o do not release harmful substances

o safe for use, particularly in food packaging.

• Establishing clear biodegradability standards to ensure complete natural degradation without harmful residues.

2) Ensuring Safety and Security

• Establishing occupational safety standards in the bioplastic production process.

• Requiring transparent labeling related to:

o material composition,

o product properties, and

o post-use disposal methods, to prevent misuse.

3) Ensuring Environmental and Ecosystem Sustainability

• Ensuring that raw materials (starch, cellulose, lignin) are sourced sustainably without:

o deforestation,

o excessive resource exploitation, or

o ecosystem disruption.

• Limiting agricultural land use for bioplastic production to a maximum of 5% of total national food-producing land.

3. Governance Actions

Governance Action 1 : Mandatory Standards for Bioplastic Production to Prevent Biological Risks

Key Actors

Government regulators, National Agency of Drug and Food Control (BPOM), Ministry of Health, Ministry of Industry.

Objective

To ensure that bioplastics produced and distributed are safe for human health, leave no harmful residues, and are genuinely biodegradable.

Design

• Establishment of national standards covering:

o toxicity testing,

o biological degradation testing,

o post-use residue testing.

• Mandatory laboratory testing and certification prior to market distribution.

• Audits and compliance enforcement by BPOM and the Ministry of Health.

• Mandatory labeling indicating certification status and estimated degradation time.

• Sanctions, ranging from fines to revocation of production licenses, for non-compliance.

Assumptions

• Mandatory standards will encourage the development of safe and fully biodegradable bioplastics.

• Strict oversight will minimize harmful residues.

• Certification costs may increase product prices and pose challenges for small and medium enterprises (SMEs).

Risks of Failure and “Success”

• Risk of failure:

o overly lenient standards may lead to greenwashing

o overly strict standards may hinder innovation and adoption.

• Risk of “success”:

o certified products may be perceived as completely safe, leading to neglect of proper post-use waste management.

Governance Action 2 : Sustainable Bioplastic Adoption and Economic Incentives

Key Actors

Government, Ministry of Finance, SMEs, manufacturing industries, Ministry of Creative Economy, Ministry of Industry, schools, universities, and the public.

Objective

Bioplastics remain relatively unfamiliar to the public. This policy aims to introduce the advantages of bioplastics while encouraging reduced use of single-use plastics. Economic incentives are provided because bioplastic production costs remain higher than fossil-based plastics and to support long-term adoption.

Design

• Development of subsidy programs, tax incentives, or research grants for bioplastic producers.

• Training and skill development programs for SMEs to support innovation in bioplastic-based products.

• Public awareness campaigns and initiatives promoting the downstream transition from single-use plastics to bioplastics.

Assumptions

• Incentives can shift industrial and market behavior.

• Government oversight can prevent misuse of incentives.

• New bioplastic-based innovations will emerge.

• Reduced consumption of single-use plastics will support sustainable adoption.

Risks of Failure and “Success”

• Risk of failure: unequal incentive distribution may widen gaps between large industries and SMEs.

• Risk of “success”: rapid production growth without adequate waste management systems may create new waste-related challenges.

Governance Action 3 : Standards for Environmentally Sustainable Raw Material Use

Key Actors

Government, Ministry of Environment and Forestry (KLHK), agricultural and food experts, manufacturing industries, environmental experts.

Objective

To prevent land exploitation, deforestation, and threats to food security arising from bioplastic raw material sourcing.

Design

• Prioritization of organic waste as raw materials.

• Establishment of maximum limits on the use of primary food crops.

• Prohibition of deforestation for raw material supply.

• Sustainability certification (zero deforestation, 5–10% crop rotation, zero hazardous waste).

• Supply chain traceability systems using blockchain technology to prevent greenwashing.

Assumptions

• Organic waste availability is sufficient and can be consistently supplied at industrial scale.

• Regulatory enforcement and traceability systems function effectively and transparently.

Risk of Failure

• Insufficient organic waste supply or weak enforcement leads to continued land exploitation.

• High compliance costs reduce industry participation, especially among small producers.

Risk of “Success”

• Increased production costs raise bioplastic prices and limit market accessibility.

• Strict standards shift unsustainable practices to informal or unregulated sectors.

4. Government governance assessment

Option 1 : Mandatory production standards that do not pose biological risks

Option 2 : Adoption of sustainable bioplastics and provision of economic incestives

Option 3 : Standards for raw material use that do not harm the environment

5. Prioritized Governance Options

Main Recommendation

A combination of Option 1 (mandatory production standards) and Option 3 (sustainable raw material standards).

Rationale

• Option 1 is critical for preventing biological and health risks.

• Option 3 ensures environmental sustainability and balance between the bioplastic industry and food security.

• Option 2 remains important but is more effective if implemented gradually to avoid excessive fiscal burden.

6. Ethical Reflection

Although these bioethical issues are not directly related to bioplastics, they are conceptually relevant as they reflect broader ethical challenges in bioengineering innovation.

Bioethical Issues • Risks to mass genetic data privacy • Gene editing risks related to off-target effects and permanent germline modifications • Inequality in access to biotechnology • Risks of GMO contamination

Governance Responses

• Establishment of an independent genetic data oversight body

• Mandatory gene-editing safety testing standards at the cellular, model organism, and bioinformatics simulation levels

• Technology transfer and capacity-building cooperation

• Long-term ecological testing prior to GMO release into the environment

Final Reflection

• Bioethics is essential because biological innovation has wide-ranging impacts beyond scientific outcomes, affecting human health, the environment, and social structures.

• Bioethics cannot be addressed from a single perspective, as innovation involves diverse interests, including researchers, governments, industries, and affected communities.

• Stakeholder collaboration is necessary to establish balanced governance, ensuring that bioethical regulations are technically sound, socially fair, and environmentally sustainable.

QUIZ PRE CLASS 2

Questions from Professor Jacobson

1. 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?

DNA polymerase has an intrinsic error rate of approximately 10⁻⁵ to 10⁻⁷ errors per nucleotide during initial DNA synthesis. With the presence of 3’→5’ exonuclease proofreading activity, the error rate is reduced to around 10⁻⁷–10⁻⁸. When combined with DNA mismatch repair mechanisms, the final error rate can be as low as ~10⁻¹⁰ errors per base per replication.

In comparison, the human genome consists of approximately 3 × 10⁹ base pairs. Without error-correction mechanisms, this would result in an unsustainable number of mutations per cell division.

Biology addresses this discrepancy through:

1).Proofreading by DNA polymerase, which corrects errors immediately during replication.

2).Mismatch repair systems, which detect and repair distortions in the DNA helix after replication.

3).Genetic redundancy and tolerance, particularly in non-coding regions of the genome.

Together, these mechanisms ensure highly accurate DNA replication despite the large size of the human genome.

2. How many different ways are there to code for an average human protein? Why don’t all of these codes work in practice?

In theory, an average human protein (~300 amino acids long) can be encoded by an astronomically large number of different DNA sequences due to the degeneracy of the genetic code, where most amino acids are encoded by multiple synonymous codons.

If each amino acid has, on average, three synonymous codons, the total number of possible coding sequences is approximately 3³⁰⁰.

However, in practice, not all of these sequences function effectively due to:

1).Codon usage bias, where organisms preferentially use specific codons.

2).mRNA stability and secondary structure, which can interfere with transcription and translation.

3).Regulatory constraints, including ribosome binding efficiency and splice-site recognition.

4).Protein folding and toxicity issues, which can arise from altered translation kinetics.

Thus, while the genetic code allows many theoretical possibilities, biological function severely constrains which coding sequences are viable.

Questions from Dr. LeProust

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

The most commonly used method for oligonucleotide synthesis today is solid-phase phosphoramidite DNA synthesis.

This method involves:

1).Sequential addition of nucleotides to a growing chain attached to a solid support

2).Highly controlled chemical reactions

3).Automation, enabling high-throughput synthesis of short DNA oligos

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

Producing oligonucleotides longer than ~200 nucleotides via direct chemical synthesis is difficult because:

1).Each coupling step has less than 100% efficiency

2).Errors such as deletions and truncations accumulate with length

3).The yield of full-length product decreases exponentially as oligo length increases

As a result, purification of accurate, full-length oligos becomes increasingly impractical beyond this length.

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

Direct synthesis of a 2000 bp gene is not feasible because:

1).The cumulative error rate would be extremely high

2).The yield of full-length product would be nearly zero

3).Purification would be technically and economically impractical

Therefore, long genes are constructed using assembly of shorter oligonucleotides through methods such as PCR-based assembly, Gibson assembly, or Golden Gate cloning.

Homework Question from George Church

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

The 10 essential amino acids in animals are:

1).Histidine

2).Isoleucine

3).Leucine

4).Lysine

5).Methionine

6).Phenylalanine

7).Threonine

8).Tryptophan

9).Valine

10).Arginine (essential in many animals, particularly during growth)

The lysine contingency refers to the fact that animals cannot synthesize lysine de novo and must obtain it from dietary or microbial sources.

This has important implications:

1. Ecosystems and food chains depend on lysine-producing organisms such as plants and microbes.

2. Lysine represents a metabolic and evolutionary bottleneck.

3. This supports Professor Church’s view that biochemical dependencies can be exploited for biocontainment, for example by engineering organisms that require an external lysine supply.

This perspective reframes the genetic code not only as an information system, but also as a tool for ecological control and biosafety.

References / AI use: Lecture 2 slides; standard molecular biology textbooks (e.g., Alberts et al., Molecular Biology of the Cell). AI (ChatGPT) was used for synthesis and conceptual explanation.

Week 2 HW: DNA Read, Write & Edit

Part 1: Benchling & In-silico Gel Art

PART 3: DNA Design Challenge

3.1 Choose your protein

Erythropoietin is a hormone that stimulates red blood cell production. Selected because:

1. Vital in anemia therapy

2. High-value biotechnology protein

3. Relevant to the pharmaceutical industry

Erythroproietin :

sp|P01588|EPO_HUMAN Erythropoietin OS=Homo sapiens OX=9606 GN=EPO PE=1 SV=1 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHC SLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQL HVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKL KLYTGEACRTGDR

3.2 Reverse Translate : Protein sequence to DNA sequence Erythroproietin DNA Sequence:

sp|P01588|EPO_HUMAN Erythropoietin OS=Homo sapiens OX=9606 GN=EPO PE=1 SV=1 ATGGGGGTGCACGAATGCCCAGCATGGTTGTGGCTACTATTGAGCCTTCTGTCCTTGCCCTTAGGTCTCCCTGTACTTGG GGCGCCCCCCCGACTAATATGTGACTCGCGGGTTTTAGAGCGGTACCTGTTGGAAGCAAAAGAAGCGGAAAATATCACTA CTGGCTGCGCTGAACATTGTTCCTTAAATGAGAATATCACAGTTCCCGACACCAAGGTAAATTTTTATGCGTGGAAACGC ATGGAGGTTGGCCAACAAGCAGTCGAAGTTTGGCAGGGGTTAGCGCTACTTTCTGAGGCAGTGCTTAGAGGCCAGGCATT GTTAGTAAATTCAAGCCAGCCTTGGGAGCCTCTACAACTTCATGTGGACAAAGCCGTGTCAGGCCTGAGATCCCTAACTA CGCTCCTCCGCGCGCTAGGAGCGCAAAAAGAGGCTATCAGTCCGCCCGACGCAGCTTCTGCCGCCCCACTCCGTACCATA ACAGCTGACACTTTCCGAAAACTTTTCAGAGTTTATTCAAACTTCCTACGAGGTAAATTGAAATTATACACTGGCGAAGC CTGCAGGACTGGGGATCGC

3.3 Codon Optimization

For this project, Escherichia coli has been chosen as the expression host.

ATGGGTGTGCACGAATGCCCAGCATGGTTGTGGCTACTGTTGAGCCTTCTGTCCTTGCCGTTAGGTCTCCCTGTACTTGGGGCGCCCCCGCGTCTTATTTGTGATTCGCGTGTTCTGGAGCGGTACCTGTTGGAAGCCAAAGAAGCGGAAAATATTACTACCGGCTGCGCTGAACATTGTTCCTTAAATGAAAACATCACAGTTCCGGACACCAAGGTCAACTTTTATGCGTGGAAACGCATGGAGGTCGGCCAACAGGCGGTCGAAGTGTGGCAGGGGCTGGCGCTACTGAGCGAGGCAGTGCTTCGTGGCCAGGCACTGTTAGTAAATAGTAGCCAGCCTTGGGAGCCGCTGCAACTGCATGTGGACAAAGCCGTGTCAGGCCTGCGCTCGCTGACGACGCTCCTCCGCGCGCTGGGAGCGCAGAAGGAAGCTATCAGTCCGCCGGATGCAGCCTCTGCCGCCCCACTGCGTACCATTACCGCTGATACATTCCGAAAACTGTTCCGTGTTTATTCAAACTTTCTGCGCGGTAAACTGAAATTATACACTGGTGAAGCCTGCAGAACGGGCGATCGC

3.4 You have a sequence! Now what?

Once the DNA sequence encoding Erythropoietin (EPO) has been designed and codon-optimized for E. coli, the next step is to produce the protein using an appropriate expression system. Two major technological approaches can be used: cell-dependent expression systems and cell-free expression systems.

A. Cell-Dependent Expression System (Using E. coli)

In this project, a cell-dependent system is used with the pET-28a expression vector.

Step 1: Cloning into pET-28a

The optimized EPO gene is inserted into the Multiple Cloning Site (MCS) of the pET-28a plasmid. The construct includes:

a. T7 promoter

b. Ribosome Binding Site (RBS)

c. Start codon (ATG)

d. EPO coding sequence

e. Stop codon

f. Optional His-tag for purification

This produces the recombinant plasmid pET-28a-EPO.

Step 2: Transformation into E. coli

The recombinant plasmid is introduced into competent E. coli cells (e.g., BL21(DE3)). Transformed cells are selected using kanamycin resistance encoded by the plasmid.

Step 3: Transcription

Upon induction:

1. The T7 RNA polymerase recognizes the T7 promoter.

2. The DNA sequence is transcribed into messenger RNA (mRNA).

3. During transcription:

   a. Adenine (A) pairs with Uracil (U)

   b. Thymine (T) in DNA becomes Uracil (U) in RNA

The result is an mRNA molecule complementary to the DNA template strand

Step 4: Translation

1. The ribosome binds to the Ribosome Binding Site (RBS).

2. Translation begins at the start codon (AUG).

3. Each codon (three nucleotides) is translated into one amino acid.

4. Transfer RNA (tRNA) delivers the corresponding amino acids.

5. The polypeptide chain elongates until a stop codon is reached.

This process follows the Central Dogma of Molecular Biology:

DNA → RNA → Protein

The final product is recombinant EPO protein, typically fused with a His-tag for purification.

B. Cell-Free Expression System (Alternative Method)

Alternatively, the EPO protein can be produced using a cell-free expression system.

In this system:

a. The DNA template is added directly to a reaction mixture.

b. The mixture contains RNA polymerase, ribosomes, tRNAs, amino acids, and necessary cofactors.

c. Transcription and translation occur in vitro (outside living cells).

Advantages:

a. Faster protein production

b. No need for cell transformation

c. Suitable for rapid screening

However, for large-scale production, cell-dependent systems are generally preferred.

3.5 [Optional] How does it work in nature/biological systems?

In human cells:

a. Alternative splicing generates isoforms.

b.RNA editing may modify nucleotides post-transcription.

c. Post-translational modifications (e.g., glycosylation in EPO) alter protein stability and function.

In contrast, E. coli:

a. Does not perform alternative splicing.

b. Does not process introns.

c. Does not perform complex glycosylation.

Thus, recombinant EPO produced in E. coli may differ structurally from native human EPO.

5.1 DNA Read

(i) What DNA would you want to sequence and why?

Primary DNA to Sequence in This Project

I would sequence:

1. The recombinant pET-28a-EPO plasmid

2. The EPO coding sequence (CDS)

3. The promoter–insert junction regions

Why?

a. To confirm that the EPO gene was inserted correctly.

b. To verify that no mutations occurred during gene synthesis or cloning.

c. To confirm the correct reading frame with the His-tag.

d. To ensure no premature stop codons or frameshifts are present.

Beyond this project, sequencing could be applied to sequencing disease-associated genes in Human health (e.g., cancer mutations).

(ii) What sequencing technology would you use and why?

For this project, I would use:

Sanger Sequencing Why?

a. The EPO gene is relatively short (~500–600 bp coding region).

b. Plasmid verification is well suited for Sanger sequencing.

c. High accuracy for single-gene validation.

d. Cost-effective for small constructs.

Classification

a. First-generation sequencing

b. Uses chain-termination chemistry

c. Produces highly accurate reads (~700–1000 bp per read)

Input: Purified plasmid DNA (pET-28a-EPO)

Preparation Steps:

1. Plasmid extraction from E. coli

2. Primer design (forward and reverse primers flanking insert)

3. PCR cycle sequencing reaction with labeled dideoxynucleotides (ddNTPs)

Essential Steps of Sanger Sequencing

1. DNA denaturation

2. Primer annealing

3. DNA polymerase extension

4. Random incorporation of fluorescently labeled ddNTPs

5. Chain termination

6. Capillary electrophoresis separation

7. Laser detection of fluorescent signals

Base Calling : Each ddNTP is labeled with a different fluorescent dye. When incorporated, elongation stops.Fragments of different lengths are separated and detected.The emitted fluorescence determines the base identity (A, T, C, or G).

Output : Chromatogram (electropherogram), DNA sequence file (.ab1 or .seq), Base quality scores

5.2 DNA Write

(i) What DNA would you want to synthesize and why?

In this project, I would synthesize:

Codon-optimized human EPO gene for E. coli

Purpose:

a. Produce recombinant EPO for research applications.

b. Study protein folding and expression optimization.

ii) What technology would you use for DNA synthesis?

Chosen Technology:

Chemical DNA synthesis followed by gene assembly & Commercial providers (e.g., gene synthesis companies) synthesize DNA de novo.

Essential Steps of DNA Synthesis

1. Oligonucleotide synthesis (phosphoramidite chemistry)

2. Assembly of short oligos into full-length gene

3. Error correction (if necessary)

4. Cloning into plasmid backbone (pET-28a)

5. Sequence verification

Limitations of DNA Synthesis

Speed: Synthesis of longer genes takes more time.

Accuracy: Errors can occur during chemical synthesis, Requires sequencing validation.

Scalability: Cost increases with gene length, Whole-genome synthesis remains complex and expensive.

However, for single-gene constructs like EPO, synthesis is efficient and practical.

5.3 DNA Edit

(i) What DNA would you want to edit and why?

In this project, I would edit:

The EPO coding sequence

Possible edits:

a. Improve solubility in E. coli

b. Reduce aggregation

c. Modify specific amino acids to increase stability

Beyond this project: i wish we can Correct disease-causing mutations in humans

(ii) What technology would you use?

For Plasmid Editing: Site-Directed Mutagenesis

For Genomic Editing: CRISPR-Cas systems

How CRISPR Edits DNA

1. Design guide RNA (gRNA) complementary to target DNA.

2. Cas enzyme binds to gRNA.

3. Complex locates target DNA.

4. Cas creates double-strand break.

5. Repair occurs via:

   a. Non-homologous end joining (NHEJ)

   b. Homology-directed repair (HDR)

Required Inputs : DNA template (target sequence), Guide RNA, Cas enzyme, Repair template (if precise edit desired), Host cells

Essential Design Steps

1. Identify target sequence.

2. Design guide RNA.

3. Check for off-target sites.

4. Prepare delivery system (plasmid or ribonucleoprotein).

5. Validate edits via sequencing.

Limitations of Editing Technologies

Efficiency: Editing efficiency may vary by cell type.

Precision: Off-target mutations may occur.

Delivery Challenges: Introducing CRISPR components into certain cells is difficult.

Ethical Considerations: Human genome editing raises significant ethical concerns.

Week 3 HW : Lab Automation

Published Paper on Opentrons Automation

The paper entitled AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots, published in the journal Synthetic Biology (2023), reports the development of AssemblyTron, a software platform that automates DNA assembly workflows using the Opentrons OT-2 liquid-handling robot. The system integrates DNA construct design (for example, from design software such as j5) with automated execution on the OT-2 to perform molecular biology procedures in a precise and standardized manner. AssemblyTron is designed to support the Design–Build–Test–Learn (DBTL) cycle in synthetic biology by automating PCR optimization, Golden Gate assembly, and in vivo assembly (IVA).

In the study, the authors demonstrated the system’s capabilities by constructing four-fragment chromoprotein plasmids and performing site-directed mutagenesis. The results showed that the assembly fidelity achieved by the automated system was comparable to that of manual methods. In addition, the use of AssemblyTron reduced the likelihood of human error, minimized the need for extensive technical training, and decreased reagent waste, while improving experimental reproducibility and throughput. Overall, this platform enables researchers to accelerate the “build” phase of the DBTL cycle and allocate more time to the design and analysis stages in synthetic biology research.

Final Project Automation Plan

I plan to automate cell-free protein synthesis (CFPS) screening for engineered fluorescent biosensors detecting environmental analytes like heavy metals, using Opentrons OT-2 integrated with Ginkgo Nebula for remote design and deployment.

Core Workflow :

A. Design Phase: Use Ginkgo Nebula to design and order biosensor genetic constructs (e.g., GFP variants fused to metal-binding domains), targeting 96 variants with randomized promoter strengths.

B. Prep and Deposition: 3D-print custom PCR tube racks (inspired by Opentrons directory) to hold construct templates; Opentrons pipets DNA/cofactors into a 96-well plate.

C. Reaction Setup: Echo transfers templates; dispense CFPS master mix (lysate, T7 RNA pol, NTPs); Multiflo adds lysate to initiate expression; PlateLoc seals; Inheco incubates at 30°C for 16h.

D. Readout: XPeel removes seal; PHERAstar measures fluorescence/excitation spectra to score sensor performance.

Example Python Pseudocode (Opentrons API) :

This script handles initial deposition; full protocol chains with temperature modules per recitation slides.

Hardware Additions : A. 3D-printed biosensor plate holder for precise alignment during imaging.

B. Remote monitoring via Opentrons App, synced to Ginkgo for iterative DBTL (e.g., top 10% sensors resynthesized).

This setup enables 100+ parallel reactions weekly, identifying hits for in vivo validation, all deployable remotely without intervention.

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project