Week 1 HW: Principles and Practices

1. First, describe a biological engineering application or tool you want to develop and why. This could be inspired by an idea for your HTGAA class project and/or something for which you are already doing in your research, or something you are just curious about. My idea: • The use of biotechnology to make pills or capsules , that reduce the glucose spike after a meal rich in carbohydrates . • My idea came from some podcasts which have as principal subject the book “Glucose revolution” by Jessie Inchauspe and also from her videos . • -I think that if the pills contains mulberry leaf extract , acetic acid , eriocitrin, the result be long term and will came faster. • The pills should be useful for everyone , diabetics , nondiabetics, prediabetics • The addition of mulberry leaf extract to sucrose resulted in a significantly lower glycemic response and insulinemic response compared to a matched placebo (sucrose alone). The change in blood glucose measurements were significantly lower at 15 min (p < 0.001), 30 min (p < 0.001), 45 min (p = 0.008), and 120 min (p < 0.001) and plasma insulin measurements were significantly lower at 15 min (p < 0.001), 30 min (p < 0.001), 45 min (p < 0.001), 60 min (p = 0.001) and 120 min (p < 0.001). The glucose iAUC (- 42%, p = 0.001), insulin iAUC (- 40%, p < 0.001), peak glucose (- 40.0%, p < 0.001) and peak insulin (- 41%, p < 0.001) from baseline were significantly lower for white mulberry leaf extract compared with the placebo. White mulberry leaf extract was well tolerated and there were no reported adverse events. • This study evaluated the potential effectiveness of different doses of Eriomin® on hyperglycemia and insulin resistance associated with other metabolic biomarkers in prediabetic individuals. Prediabetes patients (n = 103, 49 ± 10 years) were randomly divided into four parallel groups: (a) Placebo; (b) Eriomin 200 mg; (c) Eriomin 400 mg; and (d) Eriomin 800 mg. Assessment of biochemical, metabolic, inflammatory, hepatic, renal, anthropometric markers, blood pressure, and dietary parameters were performed during 12 weeks of intervention. Treatment with all doses of Eriomin (200, 400, and 800 mg) had similar effects and altered significantly the following variables: blood glucose (-5%), insulin resistance (-7%), glucose intolerance (-7%), glycated hemoglobin (-2%), glucagon (-6.5%), C-peptide (-5%), hsCRP (-12%), interleukin-6 (-13%), TNFα (-11%), lipid peroxidation (-17%), systolic blood pressure (-8%), GLP-1 (+15%), adiponectin (+19%), and antioxidant capacity (+6%). Eriomin or placebo did not influence the anthropometric and dietary variables. Short-term intervention with Eriomin, at doses of 200, 400, or 800 mg/day, benefited glycemic control, reduced systemic inflammation and oxidative stress, and reversed the prediabetic condition in 24% of the evaluated patients. these studies were taken from : https://pubmed.ncbi.nlm.nih.gov/36644880/ https://pubmed.ncbi.nlm.nih.gov/33858439/ https://pubmed.ncbi.nlm.nih.gov/31183921/ 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. Below is one example framework (developed in the context of synthetic genomics) you can choose to use or adapt, or you can develop your own. The example was developed to consider policy goals of ensuring safety and security, alongside other goals, like promoting constructive uses, but you could propose other goals for example, those relating to equity or autonomy. Main objective: -Ensuring the responsible and rigorous use of nutrition engineering technologies; 2.1 Patient safety: Testing carefully and prevention of the extracts side effects; 2.2 Equity of access: Preventing inequitable access to the pills , where only small groups of population can use them. the benefits have to be accessible for everyone , both in terms of cost and the information about them, which can convince they to buy them to improve them lifestyle and in order to enjoy carbohydrate -rich meals.

3 Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”). Try to outline a mix of actions (e.g. a new requirement/rule, incentive, or technical strategy) pursued by different “actors” (e.g. academic researchers, companies, federal regulators, law enforcement, etc). Draw upon your existing knowledge and a little additional digging, and feel free to use analogies to other domains (e.g. 3D printing, drones, financial systems, etc.).

3.1 Implementing strict regulation -Purpose :To ensure that the process is correct and the result is not harmful. -Desing: A set of rules it will be created and will stay as a contract that companies and researchers have to sign . -Assumptions: There might be resistance from industries or individuals who don’t thinks these rules are necessary for an already existing product; -Risks of Failure &Success: If the regulations are too strict , it could limit the research or the process for making them; 3.2 Establishing public funding initiatives -Purpose: To make the pills accessible for many categories of people , without lost profit. -Design: Government and international organizations would collaborate to fund nutritional research and subsidize treatments for low-income population. -Assumptions: The companies might not like the idea and the case and they won’t be interested to found such initiatives. -Risks of Failure &Success: If public funding is not sufficient , the program could fail, but if it works it will be very useful for many people. 3.3 Public education and awareness campaigns -Purpose: To make people to understand how glucose-lowering capsules work or their benefits and risks. -Desing: Some necessary things for campaigns are educative materials, like brochures and videos. Also will be making workshops or webinars with healthcare.

• Assumptions : People will engage with and trust the educational materials;
• Risks of Failure &Success: Low participation or attention from the public and well-informed users may increase demand faster than supply;

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. For this, you can choose one or more relevant audiences for your recommendation, which could range from the very local (e.g. to MIT leadership or Cambridge Mayoral Office) to the national (e.g. to President Biden or the head of a Federal Agency) to the international (e.g. to the United Nations Office of the Secretary-General, or the leadership of a multinational firm or industry consortia). These could also be one of the “actor” groups in your matrix. Based on scoring, I would prioritize Action1. Action2 and 3 are almost as supportive as the first one, but secondary measure. This is a very correct approach, but not very usual. Capsules that are safe for use with the intention of limiting possible incidents in the lab as well as in the nature. The capsules rated best for the prevention of incidents and promoting safe uses. Even if the cost is higher which leads to research struggles the results ale benefical.

Homework Questions from Professor Jacobson: [Lecture 2 slides] 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? 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 error rate of polymerase is 1:10^{6.The human genome contains approximately 3 billion base pairs(3x10}9).Without correction , this would result in aprox. 3000 errors every time a cell divides. Biologicaly after replication, the secondary protein systems scans the DNA to fix remaining discrepancies.Much of the human genome is non-coding (introns) or “dormant,” meaning many mutations occur in areas that don’t affect cell function.For an average human protein (~345 amino acids / 1036 bp), there are roughly 10^157 possible DNA sequences that could code for it, because most amino acids are represented by multiple codons. Homework Questions from Dr. LeProust: [Lecture 2 slides] What’s the most commonly used method for oligo synthesis currently? Why is it difficult to make oligos longer than 200nt via direct synthesis? Why can’t you make a 2000bp gene via direct oligo synthesis? The gold standarts is solid-phase phsphoramidite chemical syntesis. Synthesizing strands longer than 200 nucleotides is difficult due to stepwise error accumulation. Even with a 99.5% efficiency rate per step, the mathematical probability of a perfect sequence drops exponentially as length increases.By 200 bases, the yield of the “perfect” product is very low. Additionally, side reactions and physical crowding on the synthesis support hinder the process.Why 2000bp is Impossible via Direct SynthesisA 2000bp gene (4000 total nucleotides) cannot be made in one go because the cumulative error rate would result in zero functional product. Instead, scientists synthesize small “oligos” and then stitch them together using biological assembly methods. **Homework Question from George Church: [Lecture 2 slides] Choose ONE of the following three questions to answer; and please cite AI prompts or paper citations used, if any.

[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”? [Given slides #2 & 4 (AA:NA and NA:NA codes)] What code would you suggest for AA:AA interactions? [(Advanced students)] Given the one paragraph abstracts for these real 2026 grant programs sketch a response to one of them or devise one of your own:** The 10 Essential Amino AcidsThe ten amino acids that most animals (including humans) cannot produce on their own are:Arginine (R)*, Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Threonine (T), Tryptophan (W), and Valine (V).*Note: Arginine is often considered “semi-essential,” especially for growth.View of the “Lysine Contingency"The “Lysine Contingency” (from Jurassic Park) is a flawed scientific concept for two main reasons:Vertebrate Biology: No vertebrate can synthesize lysine naturally. Therefore, “knocking out” the ability for dinosaurs to make it doesn’t change anything—they were already dependent on their diet for it.Ecological Reality: In the wild, lysine is abundant in plants and other animals. If the dinosaurs escaped, they would simply eat lysine-rich food (like soy or meat), rendering the laboratory “failsafe” completely useless.

Week 2-DNA Read, Write, & Edit

title: ‘Week 2’ weight: 10

Part 1: Benchling & In-silico Gel Art

Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks. You might find Ronan’s website a helpful tool for quickly iterating on designs!

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

I didn’t have acces to a lab ,so I studied the protocol.

Part 3: DNA Design Challenge

3.1. Choose your protein.

I choose Dsup-Damage suppressor protein from Ramazzottius varieornatus-tardigrade, because Dsup is a fascinating protein, unique to extremophile tardigrades, which binds to nucleosomes and protects DNA from damage caused by radiation , desiccation and reactive oxigen species.This makes it highly relevant for applications in biotechnology, such as enhancing radiation resistance in cells for space exploration, cancer therapy, or environmental remediation. It’s also an intrinsically disordered protein, offering insights into novel DNA-protection mechanisms beyond traditional repair pathways.

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

I used uniprot to find Dusp protein amino acid sequence. uniprot-P0DOW4

This is the protein sequence:MASTHQSSTEPSSTGKSEETKKDASQGSGQDSKNVTVTKGTGSS ATSAAIVKTGGSQGKDSSTTAGSSSTQGQKFSTTPTDPKTFSSDQKEKSKSPAKEVPS GGDSKSQGDTKSQSDAKSSGQSQGQSKDSGKSSSDSSKSHSVIGAVKDVVAGAKDVAG KAVEDAPSIMHTAVDAVKNAATTVKDVASSAASTVAEKVVDAYHSVVGDKTDDKKEGE HSGDKKDDSKAGSGSGQGGDNKKSEGETSGQAESSSGNEGAAPAKGRGRGRPPAAAKG VAKGAAKGAAASKGAKSGAESSKGGEQSSGDIEMADASSKGGSDQRDSAATVGEGGAS GSEGGAKKGRGRGAGKKADAGDTSAEPPRRSSRLTSSGTGAGSAPAAAKGGAKRAASS SSTPSNAKKQATGGAGKAAATKATAAKSAASKAPQNGAGAKKKGGKAGGRKRK

I reversed translated the amino acid sequnce into nucleotide sequence.

This is the DNA sequence: atggcatcca cacaccaatc atccacagaa ccctcttcca caggtaaatc tgaggaaacg 61 aagaaagatg cttcgcaagg gagcgggcaa gactccaaga acgtaaccgt taccaaaggt 121 accggttcct ccgccacctc agctgccatt gtcaagacag gaggatccca aggcaaagat 181 tcctctacta cagcgggctc ttctagtact cagggacaga agttcagtac tacacctacc 241 gacccgaaaa ctttcagctc tgaccaaaag gagaaatcca aaagcccagc caaagaagtc 301 ccgtctggtg gcgatagtaa gtcccaaggt gacaccaagt ctcaaagcga cgccaaatct 361 tctggacaaa gtcagggcca gtctaaagac agcggcaaat catcttccga cagtagcaag 421 agtcactctg tcatcggagc tgtcaaagac gtcgttgcag gcgccaaaga tgtcgcagga 481 aaagccgtcg aggatgctcc tagcatcatg catactgcag tcgatgctgt gaagaacgca 541 gccacgactg tgaaggatgt ggcatcgtcg gctgcatcga ctgtggcgga gaaggtagtc 601 gatgcttacc acagtgtggt gggagacaag acggacgaca agaaagaggg cgagcacagc 661 ggcgacaaga aggacgactc caaagctgga agtggctctg gacaaggtgg tgacaacaag 721 aagtctgaag gagagacttc tggccaagca gaatccagct ctggcaacga aggagctgct 781 ccagccaaag gccgtggtcg tggacggcct ccagcagctg ctaaaggagt tgctaagggt 841 gctgcaaagg gcgctgccgc ctccaaagga gccaagagcg gtgctgaatc ctccaaggga 901 ggagaacagt cgtcaggaga tatcgagatg gcagatgctt cctccaaggg aggctcggac 961 cagagggatt ccgcggcgac cgttggcgaa ggtggtgcat caggcagtga gggtggagct 1021 aagaaaggca gagggcgggg cgctggtaag aaagcggatg cgggtgatac gtccgctgag 1081 ccgcctcggc ggtcgtcccg cctgacgtct tcaggtacag gggcgggttc cgctccagct 1141 gcagcgaaag gcggagcgaa gcgtgctgct tcttcctcca gtacaccttc caacgctaag 1201 aagcaagcga ctggaggtgc tggcaaagct gctgccacca aagcaactgc tgccaaatcg 1261 gcagcctcta aagctcccca gaatggcgca ggtgccaaga agaagggagg aaaggctgga 1321 ggacggaaga ggaagtaa

3.3. Codon optimization.

3.4. You have a sequence! Now what?

 To produce the Dsup protein from this DNA, technologies leveraging the central dogma (DNA → RNA → protein) can be used. I'll describe both cell-dependent and cell-free methods.

Cell-dependent (in vivo): Insert the optimized DNA into an expression vector (e.g., pET-28a with T7 promoter). Transform the plasmid into E. coli (e.g., BL21(DE3) strain) via electroporation or heat shock. Induce expression with IPTG, which activates the T7 RNA polymerase to transcribe the DNA into mRNA. The mRNA is then translated by ribosomes using tRNAs to assemble the protein. Harvest cells, lyse them, and purify Dsup (e.g., via His-tag affinity chromatography if added). Cell-free (in vitro): Use a cell-free expression system (e.g., PURExpress or wheat germ extract). Transcribe the DNA into mRNA using T7 RNA polymerase, NTPs, and buffers. Then, add the mRNA to a translation mix containing ribosomes, tRNAs, amino acids, energy sources (ATP/GTP), and cofactors. Translation occurs directly, producing the protein without cells. This is faster for screening but lower yield. Both methods allow scalable production, with cell-dependent being cheaper for large quantities.

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

Describe how a single gene codes for multiple proteins at the transcriptional level. Try aligning the DNA sequence, the transcribed RNA, and also the resulting translated Protein!!! See example below.

In nature, the Dsup gene in tardigrades is transcribed into mRNA in the nucleus under stress conditions (e.g., radiation). The mRNA is exported to the cytoplasm, where it’s translated into the Dsup protein by ribosomes. Dsup then localizes to the nucleus via its nuclear localization signal, binding nucleosomes to shield DNA. For alignment (using Benchling-style visualization): DNA: atggcatccacacaccaatcatcc… (original sequence as in 3.2) RNA: auggcauccacacaccaaucaucc… (T → U) Protein: M A S T H Q S S T E P S… (each codon triplet → 1 AA) This shows the flow: DNA transcribed to RNA (complementary, T→U), RNA translated in triplets (e.g., AUG = M start). Failures like mutations could alter this, but optimization helps overcome expression issues in heterologous systems.

Part 4: Prepare a Twist DNA Synthesis Order

**5.1 DNA Read

(i) What DNA would you want to sequence (e.g., read) and why? This could be DNA related to human health (e.g. genes related to disease research), environmental monitoring (e.g., sewage waste water, biodiversity analysis), and beyond (e.g. DNA data storage, biobank).

I would like to sequence the skin microbiome of individuals with atopic dermatitis to identify the imbalance between beneficial and harmful bacteria, which would help me in creating a personalized treatment.

(ii) What technology or technologies would you use to perform these DNA edits and why?

I would chose the third generation sequencing , because it’s essential for distinguishing between related bacterial strands in the microbiome .The essentials steps are that the DNA is passed through a nanopore and as it moves, it creates specific fluctuations in an electrical current, which are then decoded directly into the DNA sequence.The limitations are that it has a higher error rate per base compared to short-read sequencing, though high-coverage analysis successfully mitigates this. To complete this step, I must first collect and filter my raw input to remove errors. Next, categorize the remaining data into specific groups based on their shared traits. Finally, verify the results against your original requirements to ensure everything is accurate and ready for use.

5.2 DNA Write

(i) What DNA would you want to synthesize (e.g., write) and why? These could be individual genes, clusters of genes or genetic circuits, whole genomes, and beyond. As described in class thus far, applications could range from therapeutics and drug discovery (e.g., mRNA vaccines and therapies) to novel biomaterials (e.g. structural proteins), to sensors (e.g., genetic circuits for sensing and responding to inflammation, environmental stimuli, etc.), to art (DNA origamis). If possible, include the specific genetic sequence(s) of what you would like to synthesize! You will have the opportunity to actually have Twist synthesize these DNA constructs! :)

I would synthesize a Synthetic Antimicrobial Peptide (AMP) Genetic Circuit. This system uses a Quorum Sensing “switch”—a promoter that only activates when it detects high concentrations of S. aureus—linked to a gene for an Antimicrobial Peptide (like Omiganan). In patients with atopic dermatitis, the skin’s barrier is overwhelmed by harmful bacteria. By writing a circuit that only triggers in the presence of this specific pathogen, we create a personalized “search-and-destroy” therapeutic that restores microbial balance without the broad-spectrum damage of traditional antibiotics.

(ii) What technology or technologies would you use to perform this DNA synthesis and why? Also answer the following questions: What are the essential steps of your chosen sequencing methods? What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?

For the synthesis, I would use Silicon-Based DNA Synthesis (Twist Bioscience). This technology uses a silicon chip to “write” thousands of different DNA sequences simultaneously, making it more scalable and cost-effective than old-fashioned plastic plate methods.

To verify that the synthesized DNA perfectly matches my design, I would use Next-Generation Sequencing (NGS). The essential steps include Library Prep , Cluster Generation , Sequencing by Synthesis (identifying bases via fluorescent flashes), and Data Analysis (reassembling the final code). While NGS is highly scalable, its main limitations are its total speed (taking 24–48 hours) and a slight dip in accuracy when encountering homopolymers.

Week 3 HW: Opentrons art

1 Generate an artistic design using the GUI at opentrons-art.rcdonovan.com. I searched some images and when I found what I wanted I made a screenshot of it and I put it in openntrons.

Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications.

I find this article " AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots “, which is prety interesting and I choose to describe it. The article introduces AssemblyTron, an open-source Python software designed to automate the “Build” step in synthetic biology. It acts as a bond between DNA design software (like j5) and affordable laboratory robots (the Opentrons OT-2). AssemblyTron is a open-source software tool that uses a robot to build DNA automatically, making biological engineering faster, cheaper, and more accurate. It make the work in a shorter time , but with the same quality as a human and give to reaschers mor time to focus on more important things.

Week 4 HW: Protein Design Part I

A

Answer any NINE of the following questions from Shuguang Zhang: (i.e. you can select two to skip)

1. How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons)

A Dalton is equal to 1/12 atom of carbon, so aprox. 1.66x10^{-27kg. I will chose beef , because the type isn’t specified . 100g of beef has aporx. 25g protein, so 500g has 125g protein. 1g=6.022x10}23 atomic mass units =>125g=7,5275 x 10^25 Dalton

2.Why do humans eat beef but do not become a cow, eat fish but do not become fish?

    Humans eat beef but do not become a cow, eat fish but do not become fish, because these foods are decomposed in substances that are used or throw away . They don’t  have an impact on our DNA so we don’t become cows or fishes.

3. Why are there only 20 natural amino acids?

There are only 20 natural amino acids because like that they are more stable and it can provide a big variety of proteins.

4. Can you make other non-natural amino acids? Design some new amino acids.

Yes, it is possible , but I won’t do it.

5. Where did amino acids come from before enzymes that make them, and before life started?

Before enzymes that make them, and before life started amino acids come from abiotic synthesis. Simple inorganic molecules reacted under high-energy conditions.

6. If you make an α-helix using D-amino acids, what handedness (right or left) would you expect?

7. If you make an α-helix using D-amino acids , there will be a left handedness.

8. Why are most molecular helices right-handed?

9. Why do β-sheets tend to aggregate? • What is the driving force for β-sheet aggregation?

10. Why do many amyloid diseases form β-sheets? • Can you use amyloid β-sheets as materials?

11. Design a β-sheet motif that forms a well-ordered structure.

Part B: Protein Analysis and Visualization

In this part of the homework, you will be using online resources and 3D visualization software to answer questions about proteins. Pick any protein (from any organism) of your interest that has a 3D structure and answer the following questions:

1Briefly describe the protein you selected and why you selected it.

I chose the same protein that I used in homework two, because Dsup is very interseting and I like very uch tardigrades.

2 Identify the amino acid sequence of your protein.

MASTHQSSTEPSSTGKSEETKKDASQGSGQDSKNVTVTKGTGSSATSAAIVKTGGSQGKDSSTTAGSSSTQGQKFSTTPTDPKTFSSDQKEKSKSPAKEVPSGGDSKSQGDTKSQSDAKSSGQSQGQSKDSGKSSSDSSKSHSVIGAVKDVVAGAKDVAGKAVEDAPSIMHTAVDAVKNAATTVKDVASSAASTVAEKVVDAYHSVVGDKTDDKKEGEHSGDKKDDSKAGSGSGQGGDNKKSEGETSGQAESSSGNEGAAPAKGRGRGRPPAAAKGVAKGAAKGAAASKGAKSGAESSKGGEQSSGDIEMADASSKGGSDQRDSAATVGEGGASGSEGGAKKGRGRGAGKKADAGDTSAEPPRRSSRLTSSGTGAGSAPAAAKGGAKRAASSSSTPSNAKKQATGGAGKAAATKATAAKSAASKAPQNGAGAKKKGGKAGGRKRK

How long is it? What is the most frequent amino acid? You can use this Colab notebook to count the frequency of amino acids.

THe protein ha 445 aminoacids.The most frequent aminoacid is Serine. It apears 84 times.

How many protein sequence homologs are there for your protein? Hint: Use Uniprot’s BLAST tool to search for homologs.

I searched for homologs in Uniprot and I think I managed to find them . So, I opened the Uniprot link is on the htgaa website at week 4 . I paste the aminoacid sequence and then I waited it to run.

Does your protein belong to any protein family?

NO, it doesn’t belong to any protein family.

**3 Identify the structure page of your protein in RCSB

When was the structure solved? Is it a good quality structure? Good quality structure is the one with good resolution. Smaller the better (Resolution: 2.70 Å)

Are there any other molecules in the solved structure apart from protein? Does your protein belong to any structure classification family?

Nucleosome-Binding Protein

3 Open the structure of your protein in any 3D molecule visualization software: Visualize the protein as “cartoon”, “ribbon” and “ball and stick”.

Color the protein by secondary structure. Does it have more helices or sheets?

It has more helices than sheets.

Color the protein by residue type. What can you tell about the distribution of hydrophobic vs hydrophilic residues?

Most of the protein will remain green, with a few small cylinders representing transient alpha-helices.

Visualize the surface of the protein. Does it have any “holes” (aka binding pockets)?

To color by secondary structure I used color red, ss h; color yellow, ss s; color green, ss l+’'.

Week 5— Protein Design Part II

A P00441 is the unique identifier for Human Superoxide Dismutase 1.

This is the original squence: MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

This is the mutation squence: utant Sequence (Target): MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

Week 6 HW: Genetic Circuits Part I: Assembly Technologies

What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

The components in the Phusion High-Fidelity PCR Master Mix are Phusion DNA Polymerase, deoxynucleodites, reaction buffer.

-Phusion DNA Polymerase-copies the DNA

-Reaction buffer-maintains optimal biochemical proprieties

What are some factors that determine primer annealing temperature during PCR?

-specific annealing enzyme use and their optimal catalysing activity temperature;

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 amplifies DNA, while r.e.d. cut DNA. Mainly, PCR is used to get more genetic material to work with- either test or express. R.e.d. is used in DNA testing and gene insertion in Gibson assembley and others.

How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

How does the plasmid DNA enter the E. coli cells during transformation?

Electroporation or thermic shock.

Describe another assembly method in detail (such as Golden Gate Assembly)

Gibson Assembly is a cloning method that sticks together multiple PCR fragments to make a single circular plasmid, without needing traditional restriction enzymes or separate ligation steps.

Each fragment is designed so that its ends include 20–40 base‑pairs of matching sequence with the neighboring fragment, forming overlapping “sticky ends” in the right order.

A DNA polymerase fills in any gaps left after annealing, and a DNA ligase seals the nicks, giving a fully closed, double‑stranded DNA molecule. Because the reaction is isothermal and the overhangs are designed computationally, Gibson can assemble many fragments in one step and is very flexible in what kind of vector you can use.

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project