Week 1 HW: Principles and Practices

About my project.

Question 1 First, describe a biological engineering application or tool you want to develop and why.

I’d Like to Do

I would like to develop an anthocyanin-based reporter system for Solanum aethiopicus. I would like it to use native genetic elements to fit in with my desire to do ethical biotechnology that fits in my own beliefs.

Development of a Cisgenic Anthocyanin-Based Visible Reporter System in Solanum aethiopicum

Overview

Reporter systems are important for studying biotechnology and understanding how life works. In resource-strained contexts, these kinds of experiments are hard to do due to high costs and equipment needs. They traditionally use elements like fluorescent or luminescent proteins from jellyfish and fireflies. The additional problem on an ethical level is that they rely on species-mixing which is contrary to the beliefs of some religions. In this project, the aim is to develop and demonstrate the efficacy of a reporter system that does not rely on species mixing. To do this, the project will use strictly cisgenic, same species elements to build the reporter system, using Solanum aethiopicum as a novel model organism.

Question 2 Next, describe one or more governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm). Break big goals down into two or more specific sub-goals.

I would like its usage to be straight forward as a gene-edited technology that does not outcompete its wild counterparts. It contributes towards an ethical future because it allows people with limited resources to safely carry out analyses in Solanum aethiopicum to understand gene function and maybe design new varieties with better traits that match their needs. All this can be done without mixing species which matters to people who don’t want to do this because of their spiritual and personal beliefs.

Question 3 Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”).

Purpose: What is done now and what changes are you proposing? Now, gene-edited technologies such as vaccines are not treated as “genetically modified”, they are regulated by the same organisation that regulates medicines in Zambia. I think this is not a good idea. Although my technology fits this description, I don’t want to normalise and naturalise these technologies. They are still technologies that should be traced as such. option1I propose separate legislation that speaks directly to gene edited products like mine where no new DNA is introduced from another organism. Currently these products are treated as though they are natural, that does not sit well with me. I believe there should be traceability and vigilance even though it is perfectly safe when it is deployed to the public.

Design: What is needed to make it “work”? (including the actor(s) involved - who must opt-in, fund, approve, or implement, etc) The buy-in of indigenous researchers. They need to opt to use Solanum aethiopicus and my reporter system to do routine plant biotechnology investigations especially about traits that are applicable to their context such as drought tolerance or pathogen resistance. Option2The authorities (the Ministry) could make it a recommended model organism for the nation’s researchers to use. Assumptions: What could you have wrong (incorrect assumptions, uncertainties)? The gene editing could cause not unintended consequences. Maybe the anthocyanin expression may not work to a degree where phenotypes are easily visible. Option3The idea is that this technology makes it easy for researchers to see gene expression cheaply and easily. It does not work if you need high tech equipment to detect the anthocyanin expression from the reporter system. So its cost must be kept low Risks of Failure & “Success”: How might this fail, including any unintended consequences of the “success” of your proposed actions? People might not want to use a new model organisms, perhaps they want to stick to Arabidosis or Medicago for their research.

Question 4 Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals. The following is one framework but feel free to make your own:

Question 5 Last, drawing upon this scoring, describe which governance option, or combination of options, you would prioritize, and why. Outline any trade-offs you considered as well as assumptions and uncertainties. I would opt for keeping the cost of the technology low through some kind of subsidy framework and I would also look at the involvement of the Ministry in encouraging uptake of the model organism and its specially developed reporter systems

Answers to Questions

Why read, write & measure polymers? To understand and exploit natural and designed properties like chirality and reactivity How many basepairs do we need to synthesize? from less than 960 bp because that is the average bacterial protein size. There are plenty that are much smaller such as heat shock proteins and cyclotides in plants Why use a custom panel from twist? Their panels show exceptional performance across low variant allele frequencies, that is one reason.

Week 2 HW: DNA Read Write and Edit

Part 1

Part 2 Gel Art

Part 3

3.1. Choose your protein.

I’ve chosen Brick1 protein from Arabidopsis thaliana.

AEC07332.1 BRICK1 Arabidopsis thaliana MAKAGGITNAVNVGIAVQADWENREFISHISLNVRRLFEFLVQFESTTKSKLASLNEKLDLLERRLEMLE VQVSTATANPSLFAT

3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

AEC07332.1 BRICK1 Arabidopsis thaliana ATGGCTAAGGCTGGGGGGATCACCAATGCGGTAAACGTTGGAATTGCAGTCCAGGCCGATTGGGAAAATAGAGAGTTCATTAGCCACATATCCCTGAATGTTCGCCGATTATTTGAATTCCTAGTACAATTTGAGTCCACAACAAAGTCGAAGCTAGCTAGCCTCAATGAGAAGCTGGACCTGTTGGAACGTCGACTCGAAATGCTGGAAGTACAGGTCTCCACTGCCACGGCGAACCCTAGTCTGTTTGCAACG

Using https://proteiniq.io/app/protein-to-dna

3.3. Codon optimization.

Original Sequence: GC=48.63%, CAI=0.65

ATGGCTAAGGCTGGGGGGATCACCAATGCGGTAAACGTTGGAATTGCAGTCCAGGCCGATTGGGAAAATAGAGAGTTCATTAGCCACATATCCCTGAATGTTCGCCGATTATTTGAATTCCTAGTACAATTTGAGTCCACAACAAAGTCGAAGCTAGCTAGCCTCAATGAGAAGCTGGACCTGTTGGAACGTCGACTCGAAATGCTGGAAGTACAGGTCTCCACTGCCACGGCGAACCCTAGTCTGTTTGCAACG

Improved DNA: GC=58.82%, CAI=0.95

ATGGCTAAGGCCGGGGGCATCACCAACGCCGTGAATGTGGGCATCGCCGTGCAGGCCGACTGGGAAAACCGGGAGTTCATCTCCCACATTTCTCTGAACGTGAGAAGACTGTTCGAGTTCCTGGTGCAGTTCGAGAGCACCACCAAGTCCAAGCTGGCAAGCCTGAACGAGAAGCTGGACCTGCTGGAGCGGAGACTGGAAATGCTGGAGGTGCAGGTGAGCACCGCCACCGCTAACCCTTCTCTGTTCGCAACC

Benchling also has a codon optimisation tool but I used the tool found on https://en.vectorbuilder.com

3.4. You have a sequence! Now what?

This protein could be overexpressed in yeast cells and then harvested by lysing the cells. I’m not sure about cell-free methods that could be used.

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

A single gene encodes proteins as shown below. Here is the AtBRICK1 mRNA, the highlighted part is the actual part that will encode the protein. The rest are untranslated regions which carry regulatory elements amongst other things.

Week 1 Lab: Pipetting

Individual Final Project

From Zhang et al. (2015)

Bioengineered Acaricide from African Biodiversity

Moola Mutondo

Designer Cells Node

Committed Listener

Abstract

African plants contain many cyclotides, ultra‑stable peptides with major potential as drugs and biopesticides, but functional screening of this diversity is limited. The goal is to build a programmable biosensor platform to screen African plant germplasm for cyclotides that modulate a defined bacterial regulatory pathway. The project tests the hypothesis that cyclotides or related peptides which interact with LacI will cause measurable changes in a LacI‑controlled GFP reporter circuit. Specific aims are: (1) design and assemble a plasmid encoding a constitutive LacI cassette and a LacI‑regulated sfGFP reporter, (2) establish a cell‑free or E. coli assay compatible with lyophilised plant extracts, and (3) implement a small‑scale, automated screen (e.g., on an Opentrons robot) to identify extracts that reproducibly alter GFP output. Methods include DNA design and synthesis, cell‑free expression or bacterial culture, plant extract preparation, automated liquid handling, and plate‑reader fluorescence measurements.

Aim 1: Experimental Aim (this project) The first aim of my final project is to build and test a LacI–sfGFP biosensor plasmid that can report on cyclotide activity in African plant extracts by utilizing DNA design in Benchling, Twist clonal gene synthesis, and E. coli or cell‑free expression assays in multi‑well plates. I will assemble and annotate the construct (J23106–lacI cassette plus LacI‑regulated sfGFP cassette), establish reaction conditions compatible with crude extracts from lyophilised plant material, and quantify fluorescence changes using a plate reader, with the option to automate liquid handling on an Opentrons system.

Aim 2: Development Aim
The second aim is to develop a portable, cell‑free screening workflow that uses the LacI–sfGFP construct to test panels of lyophilised African plant germplasm for cyclotide activity, including in low‑infrastructure settings. This includes refining extract preparation and normalization, optimizing cell‑free assay conditions for robustness without specialized equipment, and implementing Opentrons‑based automation in the lab as a reference workflow that can be down‑scaled to simple, field‑deployable formats (e.g., pre‑aliquoted lyophilised TXTL reactions in strips or plates).

Aim 3: Visionary Aim
The third aim is to establish a distributed, field‑compatible platform that lets communities and regional labs screen local biodiversity for bioactive cyclotides using standardized cell‑free genetic circuits. In its fully realized form, this system would enable on‑site testing of plant material with minimal laboratory infrastructure, creating a networked pipeline where field screens feed into centralized follow‑up, thereby shifting peptide discovery capacity toward African institutions and the Global South.

Cell‑free systems are used so that cyclotide activity can be assayed directly from lyophilised plant material in simple, portable reaction formats, enabling on‑site testing outside fully equipped laboratories. This is directly inspired my MiniPCR BioBits products.

Problem
Africa is rich in indigenous knowledge about medicinal plants and pest control, but much of this knowledge is being lost as knowledge‑holders pass away, limiting systematic discovery of peptide leads such as cyclotides from African biodiversity.

Goal
To use African biodiversity and indigenous knowledge systems to develop and validate a cyclotide‑based peptide template for antiparasitic (e.g., acaricide) applications, starting from functional screening in a LacI–sfGFP biosensor platform.

Hypothesis
Cyclotides and related peptides discovered by combining African germplasm, indigenous knowledge, and a programmable biosensor system will yield more potent, context‑relevant, and accessible antiparasitic drug candidates than approaches that ignore local biodiversity and use patterns.

Innovation and Approach
Collect candidate cyclotide‑producing plants and ethnobotanical leads from local communities; prepare lyophilised plant germplasm and screen extracts in a LacI‑based GFP cell‑free assay (optionally automated on Opentrons) to identify bioactive peptides; integrate hits with known and predicted cyclotide structures to define peptide templates; and, in future work, use these templates for synthetic peptide design, plant transient expression, and ultimately engineered cyclotide‑producing crops.

Impact
If successful, this platform will enable African and rural labs to couple field‑compatible, cell‑free biosensing with indigenous knowledge, supporting the development of locally relevant cyclotide‑based antiparasitic interventions and, longer‑term, engineered crops that provide community‑accessible biopesticide or therapeutic functions.

SECTION 4: EXPERIMENTAL DESIGN, TECHNIQUES, TOOLS, AND TECHNOLOGY

Overview

The project will establish a LacI‑regulated sfGFP biosensor plasmid and a cell‑free, Opentrons‑assisted workflow to screen lyophilised African plant germplasm for cyclotides or related peptides that modulate LacI activity.

Experimental plan

1. Design biosensor construct in silico (1–2 days)

   • Use Benchling to design a plasmid with a standard E. coli backbone (ori, antibiotic marker) and a synthetic insert comprising two transcriptional cassettes in series:
     • Cassette 1: J23106–RBS–lacI–B0015 (constitutive LacI expression).
     • Cassette 2: Ptac/Ptrc–RBS–sfGFP–Term (LacI‑regulated GFP reporter).
   • Tools/Concepts: DNA part libraries, promoter/RBS/CDS/terminator annotation, plasmid maps.
   • Expected result: A fully annotated sequence J23106 – RBS_lacI – lacI – B0015 – Ptac/Ptrc – RBS_sfGFP – sfGFP – Term ready for synthesis and cloning.

2. Prepare Twist clonal gene order (0.5–1 day)

   • Concatenate all parts into a single insert in Benchling and export as FASTA/GenBank.
   • Submit a Twist clonal gene order specifying the insert and chosen plasmid backbone.
   • Tools: Benchling Assembly/Concatenation, Twist clonal gene ordering portal.
   • Expected result: Confirmation of a synthesis order that will deliver the complete biosensor plasmid.

3. Receive and verify biosensor plasmid (1–2 weeks turnaround; 2–3 days lab work)

   • Transform the Twist‑supplied plasmid into E. coli, plate on antibiotic, and pick colonies.
   • Verify construct by colony PCR and/or Sanger sequencing across the insert.
   • Methods: Chemical transformation, plating, miniprep, PCR, sequencing.
   • Expected result: Verified clone(s) carrying the correct LacI–sfGFP biosensor plasmid.

4. Benchmark biosensor in vivo (optional but informative; 3–5 days)

   • Express the plasmid in E. coli and measure GFP with and without IPTG (or another LacI inducer).
   • Methods: E. coli culture, inducer titration, plate‑reader fluorescence.
   • Expected result: Low baseline GFP in the absence of inducer and increased GFP upon induction, confirming functional LacI repression and dynamic range.

5. Set up cell‑free biosensor conditions (3–5 days optimization)

   • Use an E. coli TXTL or similar extract system and titrate plasmid concentration, reaction volume, and incubation time to obtain robust GFP expression and clear repression by LacI.
   • Methods: TXTL setup, small‑scale optimization in 96‑well plates, time‑course fluorescence measurement.
   • Expected result: A cell‑free condition where LacI keeps GFP low at baseline, with a defined induction window and reproducible kinetics suitable for screening.

6. Assemble lyophilised plant germplasm library (ongoing; initial batch 1–2 weeks)

   • Collect plant material guided by African indigenous knowledge and biodiversity (e.g., Rubiaceae/Violaceae and other candidate cyclotide‑producing species).
   • Lyophilise leaves, roots, or whole tissue, then mill to a fine powder and store as a germplasm library.
   • Methods: Sample collection, lyophilisation, grinding, storage.
   • Expected result: A panel of standardized lyophilised plant powders ready for extraction and screening.

7. Develop plant extract preparation protocol (3–5 days)

   • In deep‑well plates, rehydrate defined masses of lyophilised powder with extraction buffer; mix and centrifuge.
   • Transfer clarified supernatants as crude extracts; normalize by volume, dry mass, or simple absorbance.
   • Methods: Plate‑based extraction, centrifugation (off‑deck), normalization strategy.
   • Expected result: Reproducible crude extracts from each plant sample that are compatible with cell‑free reactions.

8. Program Opentrons for automated plate setup (3–7 days scripting and testing)

   • Write and debug an Opentrons protocol that:
     • Dispenses TXTL mix and biosensor plasmid into 96‑ or 384‑well plates.
     • Adds plant extracts, solvent controls, and positive controls (e.g., IPTG or known LacI ligands) to designated wells.
   • Tools: Opentrons API, labware definitions, deck layout, calibration.
   • Expected result: A reliable script that assembles complete biosensor reactions for dozens–hundreds of conditions per run.

9. Run pilot screening experiment (2–3 days)

   • Use the Opentrons‑assembled plate to test a small panel of plant extracts (e.g., 16–32 samples plus controls).
   • Incubate plates at appropriate temperature and measure GFP fluorescence over time using a plate reader.
   • Methods: Automated pipetting, plate incubation, kinetic fluorescence readout.
   • Expected result: Time‑course curves showing baseline GFP (no extract), induced GFP (positive control), and extract‑dependent deviations.

10. Define hit‑calling criteria and analyze data (2–3 days)

    • Calculate normalized GFP signals for each well relative to negative and positive controls.
    • Define thresholds for “hits” (e.g., >2‑fold increase or significant decrease compared to solvent control).
    • Tools: Python/R or spreadsheet analysis, data visualization (heatmaps, dose–response plots in later runs).
    • Expected result: A list of plant extracts that reproducibly increase or decrease GFP, indicating modulation of LacI or the reporter pathway.

11. Iterative optimization and robustness checks (ongoing; 1–2 weeks)

    • Adjust extract concentrations, reaction times, and plasmid DNA levels to reduce noise and improve discriminability between hits and non‑hits.
    • Include technical and biological replicates, and test for potential extract toxicity or general translation inhibition.
    • Methods: Design‑of‑experiments style parameter exploration, reproducibility measurements.
    • Expected result: A stable screening protocol with acceptable signal‑to‑noise and reproducible hit calls.

12. Secondary characterization of hits (future development; beyond core HTGAA timeline)

    • For top‑scoring extracts, perform dilution series and repeat TXTL assays to generate simple dose–response curves.
    • Optionally, fractionate extracts or cross‑reference with known/predicted cyclotide content from literature and in silico predictions.
    • Methods: Serial dilution, repeated TXTL assays, literature and database mining.
    • Expected result: Shortlisted plant samples and conditions that strongly suggest cyclotide or peptide modulators of LacI activity.

13. Field‑compatible format exploration (conceptual or pilot; 3–5 days if attempted)

    • Evaluate whether lyophilised or pre‑aliquoted cell‑free reagents with the plasmid can be used in simpler, low‑infrastructure formats (e.g., strips or small tubes) for non‑Opentrons, field‑adjacent testing.
    • Expected result: A proof‑of‑concept or design concept for field‑deployable assays that extend beyond the core laboratory setup.

Expected overall outcome

If successful, this experimental plan will yield:

• A verified LacI–sfGFP biosensor plasmid and robust cell‑free assay.
• An Opentrons‑enabled workflow to test many lyophilised plant extracts in parallel.
• A first set of “hit” germplasm samples whose extracts modulate LacI‑controlled GFP, forming the basis for future cyclotide identification, structural work, and peptide template design.

Techniques relevant to my project

Key (✔ = relevant / used, ✖ = not central right now).

Core lab skills

• ✔ Pipetting
• ✔ Lab Safety
• ✔ Bioethical Considerations (must check this box)

DNA / sequence work

• ✖ DNA Gel Art
• ✔ DNA Sequencing (to verify the Twist plasmid)
• ✔ DNA Editing (in silico design/edits in Benchling)
• ✔ DNA Construct Design
• ✖ Restriction Enzyme Digestion (not essential if using Twist + Gibson‑free workflows)
• ✖ Gel Electrophoresis (optional for QC)
• ✖ DNA Purification From Gel
• ✔ Databases (GenBank / NCBI for promoters, lacI, sfGFP, cyclotides)

Automation and robotics

• ✔ Lab Automation
• ✔ Creating Code for Laboratory Automation
• ✔ Using Liquid Handling Robots (Opentrons)
• ✔ Designing a Twist Order
• ✖ Creating a plan to use the Autonomous lab at Ginkgo Bioworks

Protein / design tools

• ✖ Protein Design (core project is regulatory, not de novo protein design)
• ✖ Use of Boltz or PepMLM
• ✖ Use of Asimov Kernel
• ✔ Use of Benchling
• ✔ Models and Notebooks (for data analysis / planning)
• ✔ Databases (for cyclotides, natural products)

Bioproduction / bacteria

• ✔ Chassis Selection (e.g., DH5α or similar for plasmid propagation)
• ✔ Registry of Standard Biological Parts (Anderson promoters, RBS, B0015)
• ✔ Plasmid Preparation
• ✔ Bacterial Culturing (to grow and prep the construct)
• ✔ Quality Control/Analysis (OD, fluorescence, sequencing)
• ✔ Bacterial Processing (centrifugation, lysis/DNA prep)

Cell‑free systems

• ✔ Cell Free Reactions (TXTL biosensor assay)
• ✔ Freeze-Dried Cell Free Systems (this is part of my testing and field‑deployable vision)
• ✖ miniPCR Tools (not required here)
• ✖ Protein Purification (not needed for the screen)

Cloning / assembly

• ✔ Primer Design or Selection (PCR‑based edits/QC)
• ✖ Gibson Assembly (can be optional if Twist delivery of the full plasmid is not possible)
• ✔ Other Cloning Methods (basic transformation, maybe RE‑based subcloning if needed)

Genome editing

• ✖ CRISPR/Cas9
• ✖ Designing Prime Editing gRNA

If it is decided to explicitly re‑clone or modify the plasmid by hand rather than rely entirely on Twist, then “Restriction Enzyme Digestion,” “Gel Electrophoresis,” and “Gibson Assembly” would be checked as well.

Notes about two techniques used in this study

1. Cell‑Free Reactions / Freeze‑Dried Cell‑Free Systems
I will use E. coli extract–based cell‑free reactions as the main testbed for the LacI–sfGFP biosensor, allowing direct GFP measurement from DNA without transformation. In the lab, I will tune plasmid concentration and reaction conditions to achieve a low GFP baseline (LacI repression) and a clear induced state that cyclotides can modulate. Normalized plant extracts from lyophilised African germplasm will then be added to these reactions, and changes in fluorescence will indicate effects on the LacI pathway.

The construct can be used in its linear form:

OR cloned into pUC18 (for increased stability and expression) using these primers:

Forward: GCGC | GAATTC | TTTACGGCTAGCTCAGTCCTA clamp EcoRI cassette-specific sequence

Reverse: GCGC | AAGCTT | TATAAACGCAGAAAGGCCCAC clamp HindIII reverse-complement of cassette end

2. Using Liquid Handling Robots (Opentrons) / Automation Code
I will use an Opentrons robot to assemble multi‑well plates, automating pipetting of TXTL mix, biosensor plasmid, plant extracts, and controls. A Python‑based protocol will define deck layout, labware, volumes, and well mapping so each extract and control is dispensed consistently. This automation improves reproducibility, enables larger germplasm screens, and links fluorescence readouts directly to specific plant samples.

How To Grow (Almost) Anything Industry Council companies which are associated with my final project

• Addgene
• Millipore Sigma
• New England Biolabs
• Opentrons
• Twist Biosciences

Section 5 Results and quantitative expectations

What aspect would I validate?

I would choose to validate the LacI–sfGFP biosensor design and its basic function in a cell‑free reaction. If successful, this would show that the J23106–lacI–Ptac/Ptrc–sfGFP construct can give low baseline GFP and higher GFP with an inducer, establishing a usable dynamic range for later cyclotide screens.

How I would validate it

1. I would design the biosensor plasmid in Benchling with two cassettes on a standard E. coli backbone: J23106–RBS–lacI–B0015 and Ptac/Ptrc–RBS–sfGFP–Term.
2. I would export the insert, place a Twist clonal gene order, then transform the plasmid into E. coli, plate, and miniprep candidate clones.
3. I would verify correct assembly by Sanger sequencing across key junctions.
4. I would then set up small E. coli TXTL reactions in a 96‑well plate with a fixed plasmid concentration, add either no inducer or IPTG, incubate, and record GFP fluorescence over time in a plate reader.

Techniques I would use

In this validation, I would apply DNA construct design and in silico editing to assemble the LacI–sfGFP plasmid in Benchling. I would also use basic bioproduction techniques, including bacterial transformation, plasmid preparation, and Sanger sequencing for verification. For functional testing, I would use cell‑free reactions (TXTL) to measure GFP expression directly from the plasmid. Optionally, I might also use basic data analysis / modeling (e.g., in a notebook) to quantify fold‑change and compare time courses between induced and uninduced reactions.

Data and quantitative expectations

If I performed this experiment, I would expect low baseline GFP fluorescence in the absence of inducer and roughly a 2–10‑fold increase in endpoint fluorescence in the presence of IPTG at the same DNA concentration. I would anticipate GFP time courses where induced wells rise to a higher plateau than uninduced wells over 4–8 hours, visible as separated curves or as bar plots of normalized endpoint values.

Challenges and limitations (anticipated)

I would anticipate that tuning plasmid concentration in TXTL could be tricky: too much LacI might over‑repress GFP, while too little might give high background and low dynamic range. I would plan to test several DNA concentrations and, if necessary, adjust promoter strength or separate LacI and GFP onto two plasmids. I would also expect that some future plant extracts could nonspecifically inhibit TXTL, so I would design dilution series and toxicity controls to distinguish general inhibition from true LacI‑specific effects. Finally, I would consider plate‑reader sensitivity and background fluorescence as potential issues and would plan to calibrate gain settings or use GFP standards if needed.

References

Huang et al. (2018) Huang, A., Nguyen, P. Q., Stark, J. C., Long, M., Padir, A., & Lu, T. K. (2018). BioBits™ Bright: A fluorescent synthetic biology education kit. Science Advances, 4(8), Article eaat5107. https://doi.org/10.1126/sciadv.aat5107

Nguyen et al. (2019) Nguyen, P. Q., Botyanszki, Z., Huang, A., & Lu, T. K. (2019). BioBits Explorer: A modular synthetic biology education kit. Science Advances, 4(8), Article eaat5105. (Companion to Silver et al., 2018)

Stark et al. (2023) Stark, J. C., Belter, A. M., Lakshmikanth, R., & Lu, T. K. (2023). At-home, cell-free synthetic biology education modules for remote learning. Synthetic Biology, 8(1), ysad012. https://doi.org/10.1093/synbio/ysad012

Tran et al. (2024) Tran, G. H., Tran, T. H., Pham, S. H., Xuan, H. L., & Dang, T. T. (2024). Cyclotides: The next generation in biopesticide development for eco-friendly agriculture. Journal of Peptide Science, 30(6), Article e3570. https://doi.org/10.1002/psc.3570

Sun et al. (2013) Sun, Z. Z., Hayes, C. A., Shin, J., Caschera, F., Murray, R. M., & Noireaux, V. (2013). Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. Journal of visualized experiments : JoVE, (79), e50762. https://doi.org/10.3791/50762

Zhang et al. (2015) Zhang, J., Hua, Z., Huang, Z., Chen, Q., Long, Q., Craik, D. J., Baker, A. J. M., Shu, W., & Liao, B. (2015). Two blast-independent tools, CyPerl and CyExcel, for harvesting hundreds of novel cyclotides and analogues from plant genomes and protein databases. Planta, 241(4), 929–940. https://doi.org/10.1007/s00425-014-2229-5

Supply list & budget

Core molecular biology and DNA

• Twist clonal gene synthesis (LacI–sfGFP plasmid, 1–2 kb insert) – $400
• Chemically competent E. coli (for cloning/propagation, 20–50 reactions) – $150
• Plasmid miniprep kit (50–100 preps) – $150
• Sanger sequencing (4–8 reactions) – $120
• Antibiotics (e.g., chloramphenicol, Kanamycin for alternative plasmids- small bottle) – $50
• Agar, LB/2xYT media, plates and broth – $100

Cell‑free systems and reagents

• E. coli TXTL or similar cell‑free expression kit (e.g., 24–50 reactions) – $350
• IPTG or other LacI inducer (small vial) – $50
• 96‑ or 384‑well plates (clear or black, flat‑bottom, 50–100 plates) – $150
• Plate seals (adhesive or heat seals) – $50

Plant material and sample prep

• Lyophiliser access (available at the Copperbelt University BU to prep samples before sending to the Node) – $100 (shared core vs. fee)
• Sample tubes, deep‑well plates, grinding beads or small mill – $200
• Basic extraction buffers and solvents (e.g., aqueous buffer, small volumes of MeOH/EtOH) – $150

Automation and equipment

• Opentrons OT‑2 or Flex access – $500 (Node‑dependent)
• Pipette tips compatible with Opentrons (filter tips, racks, ~5–10 boxes) – $200
• Benchtop plate reader with fluorescence (Node-dependent) – $200

General lab consumables and safety

• Pipette tips (manual pipetting; 1–2 cases mixed sizes) – $200
• Microcentrifuge tubes, 15/50 mL conical tubes – $150
• Gloves, lab coats, disinfectant, basic PPE – $100

Approximate total -$4050.-

Group Final Project