Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
Class Assignment Describe a biological engineering application or tool you want to develop and why. Aplication title: De novo design of proteins binders for neutralizing Bothrops venom toxins Antivenoms are a mix of immunoglobulins produced traditionally by the hyperimmunization of large animals with crude venom obtained from clinically-relevant snakes (Ratanabangkoon, K., 2023). Novel alternatives have emerged to neutralize venom toxins without the use of animals. For example, Torres and collaborators (2025) designed proteins with high affinity for important regions of cytotoxins from the 3FTx family. These proteins showed great neutralizating capacity in vitro and great protective capacity in vivo .
Week 2 HW: DNA Read, Write & Edit
Part 1: Benchling & In-silico Gel Art Lambda Sequence: Sequence from E.coli I cl857 S7 lambda bateriophage (Daniels, et al., 1983) available at New England Biolabs (N3011) A digest simulation was performed using the lambda sequence and 7 different restriction enzyme (EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI, and SalI). The range of fragments obtained from this simulation varies depending on the enzyme used.
Opentrons Artwork: Gel Designing Design: Snake Trimeresurus puniceus Inspired from a snake photo taken in the Oswaldo Meneses serpentarium, Lima, Peru. Art created Donovan’s Automation art interface Python Script Design Opentrons script was created following the instructions and ideas offered by the HTGAA Opentrons Colab. To create the script first I created a pseudocode with the idea of how the robot will work
Week 4 HW: Protein Design Part 1
Part A: Conceptual Questions How many molecules of amino acids do you take with a 500 grams of meat? (on average an amino acid is ~100 Daltons) Assuming whole composition of meat is protein, the number of amino acids molecules in 500 grams is 3.011 x 1024 molecules. Why do humans eat beef but do not become a cow, eat fish but do not become fish? This is because our digestion breaks down macromolecules into their monomers. Proteins are broken down into amino acids that later are used for the biosynthesis of proteins. The phenotypic characteristic of an organism is defined in its principally by its genome, and it’s not affected by the food they consume.
Week 5 HW: Protein Design Part II
Part 1: SOD 1 Binder Peptide Design Superoxide dismutase 1 sequence was retrieved from Uniprot database (P00441), this protein has a length of 154 amino acids. SOD1 Sequence: sp|P00441|SODC_HUMAN Superoxide dismutase [Cu-Zn] OS=Homo sapiens OX=9606 GN=SOD1 PE=1 SV=2 MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ The mutated version of the human SOD1 caused by an A4V mutation was retrieved from the PDB database that contains a structure obtained from an X-Ray Diffraction study with a resolution of 1.90 Å (Hough et al., 2004)
Week 6 HW: Genetic Circuits Part 1
Part 1: DNA Assembly What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion High-Fifelity PCR Master Mix offered by New England Biolabs is a product that contains a DNA polymerase with high fidelity useful for cloning and amplification of difficult amplicons. Phusion DNA polymerase contains proofreading activity (3’ -> 5’ exonuclease) and a higher fidelity 50X greater that Taq polymerase. Thermo Fisher Scientific Phusion High-Fidelity PCR Marter Mix is composed of a HF Buffer or GC Buffer; both buffers are used to reduce the error rate of the DNA polymerase. HF Buffer contains a lower error rate (4.4 x 107) than GC Buffer (9.5 x 107), however GC buffer can improve the performance of the polymerase on some difficult or long templates with high GC-rich templates or with secondary structures. The master mix is also provided with a optimized concentration of MgCl2 which is an essential cofactor that stabilizes the DNA double helix and facilitates primer annealing. High Fidelity PCR reaction can also include DMSO that is used to reduce the melting temperature through its association with Cytosine residues that changes the conformation of the DNA template. What are some factors that determine primer annealing temperature during PCR? Primer annealing temperature is affected by the Primer melting temperature, annealing temperature is usually 3-5 °C below the primer Tm this temperatura depends on the GC content of the primers or primer length and the prescense of secondary structures. Another factor that can modify the Ta is the MgCl2 concentration indicating the higher concentrations can increase the Ta. There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other. PCR is a laboratory method often used to increase the amount of a desired DNA sequences, it uses primer to determine the boundaries of the fragment and uses a DNA polymerase that can be genetically engineered to get different results. PCR is often used to amplify sequences for other analysis like sequencing or cloning. Resctriction enzyme digest used restriction endonucleases that recognize specific palindromic sequences and cuts these sites, it doesn’t not amplify the target sequence and is often used for its integration in a cloning vector for recombinant technologies. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? To ensure that the DNA sequences will be appropiate for Gibson cloning the regions must have overlaping regions that can be used to connect correctly with the vector. The digested vector mus be linear and it must be purified to prevent re-ligation with the vector. How does the plasmid DNA enter the E. coli cells during transformation? The era multiple transfection methods use to insert a target DNA sequence inside the E. coli cells different methods can be used, one method called electroporation uses an electric shock to disturb the membrane and insert the DNA vector, another method uses a heat shock to disturb and insert the desired vector. Describe another assembly method in detail (such as Golden Gate Assembly) Golde Gate Assembly uses Type IIS restriction enzyme and T4 DNA ligase that cuts outside their recognition sequence, this allows to desing overhangs that can be ligated later, later this recognition sites are removed during assembly. Golden gate method allows assembling up to nine fragments at a time in a recipient plasmid. This can be done in a single tube that contains the donors, the recipient vector, a type IIS restriction enzyme and ligase (Engler & Marillonnet,. 2013) Some advantages of the Golden Gate Assembly are:
Week 7 HW: Genetic Circuits Part II
Genetic Circuits Part II: Neuromorphic Circuits Part 1: Intracellular Artificial Neural Networks (IANNs) What advantages do IANNs have over traditional genetic circuits, whose input/output behaviors are Boolean functions? One characteristic of IANNs is that they can process continuous inputs rather than only discrete boolean data, this resembles with the natural process of a living system that is composed of a complex network of molecular interactions. Boolean login can simplify complex living activities making it difficult to process and engineer systems with this logic but IANNs uses complex analysis that can be used to reproduce complex process in living beings that uses millions of parameters. By training an IANN is possible to simulate complex genetic activities that resemble more to the living processes than only using boolean functions (Nilsson et al, 2022) Because IANNs works with continous data they can work better where a system have more molecular noise and context effect thant Boolean switches making them useful in therapetic context where there is a high amount of molecular noise that must be processed to produce a suitable answer (Müller et al., 2025)
Part A: General and Lecturer-Specific Questions General Homework Questions 1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production: Cell-free protein synthesis (CFPS) has greater flexibiltiy and experimental control that recombinar expression systems because the entire reaction happens outside a living organism. Without the use of a cell system to express the protein we can control and optimize every component of the system, making them scalable a can be applied in low amount making them suitable to analyze multiple protein candidates in parallel.
Week 10 HW: Imaging and Measurement
Homework: Final Project Figure 1 below presents some key aspects of my final project that require experimental testing and quantitative evaluation. These aspects refer to the expression of the protein binders generated using a Deep Learning Model and selected after in silico prediction of their therapeutic characteristics. Figure 1: Experimental aspects of the final project: AI-driven Antivenom: A Generative Pipeline for De novo Neutralizing Peptides Against Snake Toxins. Image generated using: Copilot AI Aspect 1: Cell-Free Expression System (CFS) Cell-Free Expression System (CFS) enables rapid expression of multiple protein variants in parallel. Since this project aims to predict and express different protein binders, CFS provides a scalable and automatable platform that avoids cell culture and allows preliminary functional testing without full purification (Cui et al., 2022) To ensure reproducibility, the CFS workflow must be standarized and quantitatively monitored. Expression efficiency will be measures by using detection tags like biotinylated lysine or His-tag into the peptide of interest, enabling detection through SDS-PAGE followed by Western Blotting. A colorimetric readout using biotin-binding secondary antibody and chromogenic substrate will aloow quantification of expression levels (Hunt et al., 2024)
Week 11 HW: Bioproduction and Cloud Labs
Part A: Artwork Canvas Contibution: I contribute with 52 dots making the “Love” Desing at the bottom left plate. I liked the collaborative working of the canvas and the constant organic evolution of the design that resembles very well with the development of synthetic biology. For a next collaborative art experiment I could propose an experiment that serves as a competence of different groups, or a collaborative project that shows thet location of the participant in the world.