Greetings! I am Rahul Yaji, from Karnataka, India. My interest in extraterrestrial habitation drew me towards biotechnnology and I completed my graduation from NMAM Institute of Technology, with a major in biotechnology engineering. My curriculum was a mix of biosciences as well as engineering topics on bioreactor design, modelling reactions and simulations. My areas of interest include synthetic biology, metabolic engineering, and biomanufacturing.
A biological engineering application or tool I want to develop and why:
I want to develop an engineered consortium of microorganisms for pilot-scale biomanufacturing on Mars. The microbes will be engineered for self-sufficent surival subject to the multitude of constraints of the red planet. This insitu resource utilization (ISRU) will be a key step towards the goal of the eventual colonization of Mars, by reducing the import from Earth. The current methods of ISRU, although in their rudimentary stages, rely on high energy chemical conversion process. My application aims at providing an alternative to this, and pave way for sustainable biomanufacturing away from the Earth.
Part 1: Benchling & In-silico Gel Art 1.1 Restriction Digestion Simulation in Benchling: 1.2 DNA Gel Art Using Automation Art: Part 2: Laboratory Work on Gel Electrophoresis Skipped due to lack of access to lab.
Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications. The paper: Slowpoke:An Automated Golden Gate Cloning Workflow for Opentrons OT‑2 and Flex
Subsections of Homework
Week 1 HW: Principles and Practices
1. A biological engineering application or tool I want to develop and why: I want to develop an engineered consortium of microorganisms for pilot-scale biomanufacturing on Mars. The microbes will be engineered for self-sufficent surival subject to the multitude of constraints of the red planet.
This insitu resource utilization (ISRU) will be a key step towards the goal of the eventual colonization of Mars, by reducing the import from Earth. The current methods of ISRU, although in their rudimentary stages, rely on high energy chemical conversion process. My application aims at providing an alternative to this, and pave way for sustainable biomanufacturing away from the Earth.
2. Governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm):
Goal 1- Prevention of forward contamination: Great care must be ensured in making sure that only the right microorganisms will colonize the desired niche. Since this may very well fit into the definition of ‘forward contamination,’ a thorough conformation of the non-existence of native Martian microbes shall guide the policy decision. International collaboration is going to be of prominence, because this goal will be of no consequence if even one of the space-capable nation refuses to abide by this. Goal 2- Address dual use concerns: It is inevitable that any microbe that has been engineered to tolerate Martian conditions would have multiple survival mechanisms that grant it an upper hand over its Earthen coutnerparts.Therefore, any type of microorganism that may even remotely prove to be pathogenic to humans must be avoided at all costs. Goal 3- Level playing fields: Monopolies and oligopolies should be prevented to the largest possible extent, especially in the early days of the settlement plan. If such imbalanced playing fields get established, it will stiffle innovations for generations to come by restricting know-hows and resources.
3. Next, describe at least three different potential governance “actions” by considering the four aspects below: 3.1 Technical Goverenance: The policy must ensure that whoever wants to set up biomanufacturing on Mars has suitably demonstrated the presence of kill switches (auxotrophic, toxin-anti-toxin etc.) to prevent accidental release into the environment. Completely orthogonal biological systems may be used in place of kill switches, but given today’s biotechnology, the former is more likely than the latter. Purpose: To prevent forward contamination. Design: Genetic circuits can be embedded with toxic-anti-toxic systems like CcdB-CcdA, MazF-MazE, and hok-soc etc. Strains auxotrophic for Glucosamine-6-phosphate Synthase ((\Delta glmS)) can be used as auxotrophic chassis organisms. Assumptions: The assumptions here would be that the strain will not bypass these kill-switches by any means, and also these kill-switches will not interefere with the organisms’ ability to synthesize the product of interest. Risks of Failure & Success: Failure to meet these parameters may lead to forward contamination, preventing the study of ‘pristine’ Martian grounds. However, the success in this context would not be permanent and require repeated peroidic demonstrations. There is also the possibility of false trigerring of kill-switch, leading to a wasted batch of products.
3.2 Regulatory Governance: A system to inventory and track all the organisms, genetic components, and manufacturing methods becomes important. This will provide a starting point to study the evolution of the microorganisms that might arise in the future. A high degree of match to the inventoried parts can help rule out any fasle-positivity regarding native Martian microbe claims. Purpose: To track any suspicious new microbes in the vicinity and beyond. Design: A robust inventory software, and the adherence of the players to documentation. Assumptions: All the players will abide by the regulations, and will not send any undocumented organisms to gain a competitive edge. Risks of Failure & Success: Failure would mean lots of undocumented and potentially unsafe microorganisms on Mars. It would also prevent any means of studying weather Mars had evolved any microbes independent of the Earth. On the other hand, a policy that is too transparent will hinder intellectual property safeguard.
3.3 Economic Incentive Governance: For this, I envision a system of “Biosecurity Bonds.” Any entity that wants to carry out biotechnology research on Mars would need to furnish a bond of a certain amount (probably in millions of dollars). If, after a period of time, no contamination can be established, the amount is refuned. If any contamination is found, the bonded amount can be utilized to ameliorate the spread. Purpose: To incentivize players to adhere to high standards of biosecurity. Design: A techno-legal framework in the form of an international treaty or agreement, among all the spacce-faring nations and also similar incentives at national level. Assumptions: None of the players will take this bond as an opportunity to “pay to pollute” and think that forfeiting the bond amount is cheaper than adhereing to the standards of biosecurity. Risks of Failure & Success: Failure can lead to an incentiveless, haphazard business models, that would aim towards establishing monopolies for profit. If this aspect is successfully governed, then there is still the risk of wealthy corporations outcompeting the not-so-wealthy ones.
4. Score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against the rubric of policy goals.
Does the option:
Option 1
Option 2
Option 3
Planetary Protection (forward)
1
3
3
• By preventing incidents
1
2
1
• By helping respond
1
3
1
Biosafety
1
3
1
• By preventing incident
1
3
1
• By helping respond
1
3
1
Redundancy and backup plans
1
2
1
• By preventing incidents
1
3
2
• By helping respond
3
1
1
Other considerations
• Minimizing costs and burdens to stakeholders
2
3
3
• Economic feasibility
3
2
3
• Not impede research
2
3
3
• Promote constructive applications
1
1
1
5. 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. Based on these parameters, I would priortize option 1, i.e, Technical Governance, and option 2, Economic Incentive Governance. Both of these would go hand in hand to cover the technical and the financial safeguards agianst the forward contamination, establishment of monopolies, and an imbalanced playing fields. However, the main trade off in not prioritizing option 2, i.e., regulatory governing would be the existence of loopholes to evade accountability. The uncertainty of non-adhering players will always remain as a looming threat in establishing a stable policy towards extraterrestrial resource utilization.
Homework questions from Dr. LeProust:
What’s the most commonly used method for oligo synthesis currently? Solid phase phosphoaramidite method is the most widely used method to synthesize oligonucleotides. Nucleoside phosphoramidites are used as the precursor molecules. It proceeds through 4 steps:
Detritylation: dimethoxytrityl group is removed from the 5’ end of the last nucleotide attached to the support using triacetic acid, activating the -OH group.
Coupling: Phosphoaramidite monomers are added along with an activator (usualy tetrazole), that protanates the phosphoaramidite. Now, the 5’ hydroxyl end of the growing chain can form a phosphite triester linkage at the 3’ phosphorous.
Oxidation: The unstable phosphite triester linkage is oxidized using iodine solution form a stable phosphate triester bond.
Capping: Once the required number of nucleotides have been synthesised using the above 3 steps, the unreacted 5’ ends are capped using an acetylation mix of acetic anhydride and N-methylimidazole. This is done to prevent wrong reactions in further cycles.
Why is it difficult to make oligos longer than 200nt via direct synthesis? If oligos are synthesized using phosphoaramidite method, the yield follows the equation Y = $C^{n}$; where Y is the yield %, C is the coupling efficiency, and n is the number of couplings. A diakósiamer (200mer) will have 199 couplings. This implies, even with a success rate of 99%, the yield would be $0.99^{199}$, which is around 13.5%. The rest of the sequences would be truncated at random lenghts less than 200 bps.
Why can’t you make a 2000bp gene via direct oligo synthesis? Using the same equation as above, we get the yield of only 1.88 * $10^{-7}$ percent, which is as good probability as nil in order to synthesize a 2 kb gene.
Homework Questions from Professor Jacobson:
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? DNA polymerarases are accurate with upto $10^{−6}$ mutations/bp. Since human genome is around 3.2 * $10^{9}$ bp long, it would imply 3200 mutations per generation. Biology deals with this descrepancy by having a multitude of proofreading mechanisms like 3’-5’exonuclease activity in the polymerase that cleaves incorrect nucleotides, mismatch repair post replication where a protein complexes can recognize the template strand and the newly-synthesized strand due to the presence of nicks in the latter, and cleave the ‘wrong’ base pairs. Then, DNA ligase joins the correct nulceotides.
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? Considering an average protein to be 375 amino acids long, and each amino acid requiring 3 codons, there can be $3^{375}$ DNA sequences for an average protein. But in reality, the number of translatable codon is limited by the properties of mRNA and the availability of tRNA. Certain DNA sequenes transcript into an mRNA that will have haripin loop, tendency to form dsRNA and other difficult-to-translate structures. And also, the translational machinery possesses a limited number of tRNA, which is the limiting factor for the number of amino acids that can be translated, and thus protein that can be synthesized.
Homework Questions from George Church:
What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”? The nutritionally essential amino acids in all animals are:
Cystine, Leucine, Lysine, Methionine, Histidine, Phenylalanin, Tyrosine, Threonine, Tryptophan, and Valine. Since lysine is already an essential amino acid, meaning, it cannot be synthesized by reptiles on their own, lysine contingency does not make any sense. It can be easily obtained by feeding on the plant matter, and the orgnaisms that feed on the plant matter, readily. The scientists of the Jurassic Park were better off in making the dinosaurs auxtorphic to certain enzymes that are very much necessary for metabolic reactions.
AJ006456.2 Acidithiobacillus ferrooxidans cyc1, cyc2, coxA, coxB, coxC, coxD and rus genes and open reading frame
TTGGCATGTCGATTTTTGGACCTCTAGTGATCACGGCCTATAATTAAACGGCATGGTTAACATGATAAAATAACGTTAGCACATAATTCTTTTCTTATGTTCGTTATTTACTTTATTGCATTTTACTGGATCGATATTCTGGCAACTATGCGCAAAATATTGATTATAAAAGCATTATAGTTATGACCATCGAGGCGATCGCGAGATGCATGGATGAGGTAGCCATGCATTTTAATGAGCGCATAAAAAGATGTTGCAAAGCATCGCGGTTTGTATTAAATAGAACGTGTGGGTATTGTTAACAACGCAACAACATTGGTTAAAGGTCGAGGCTAATTGGCATCGCGTTGTTGTGGTTTGGTGTTACCAGCCTGGCAGGAAGACCGGGCGCATGAGCGTATTTTGTTTATCTAATATGCCTGAAAGCGCATACCGCTATGGAGGGGGTTATGGTGTCATCGTCCGTTGGTTTTAAAAAGAAAAGGTTGATCGTAGCATTAGCAGCAGTTGGTGGAATGGCGTTATCTTCCAGTGCCTGGGCACTGCCATCCTTTGCGCGCCAGACCGGTTGGTCGTGCGCCGCCTGTCACACATCCTACCCGCAGTTGACGCCCATGGGCAGAATGTTCAAATTGCTCGGGTTCACGACCACAAACCTGCAACGGCAGCAGAAGCTCCAAGCCAAGTTCGGGAACAGCGTCGGTCTGCTCATATCCCGCGTGTCACAATTTTCTATCTTCCTGCAGGCCTCGGCGACCAATGTTGGTGGCGGGCAGGCGGTGTTTGGTTCTGGTAACTCTAATGCGAATGCTTCTCCCAACAATAATGTTCAGTTTCCACAACAGGTGAGCTTGTTCTATGCCGGTGAAATCACTCCGCATATCGGCTCGTTTCTGCATATCACCTACTCGGGCGGCGGCAGTGGTACCGGCGGCGGAGGATTTAGTTTTGACGACTCCAGCATTGTCTGGGCCCATCCATGGAAGTTGGGCACCAACAATCTTTTGGTTACGGGCGTAGACGTCAACAATACCCCGACTGCTATGGACTTGTGGAATACCACACCTGATTGGCAGGCACCATTTTTCTCCTCGGATTATTCGTCTTGGGGCCACGTACCTCAGCCATTCATTGAAAGTTCAGCGGGCGCGGGTTACCCATTAGCGGGTGTTGGTGTCTATGGGGCGGATATTTTTGGGCCAAACCGGGCAAACTGGCTGTACGCAGACGCCGATGTCTATACCAACGGTCAAGGAACCCAAGTCAACCCGGTTGGCGGTTTTACTGCAGCTGGCCCCCAGGGCAGGCTTTCAGGGGGCGCTCCTTATGTTCGTCTTGCCTATCAGCACGATTGGGGTGACTGGAACTGGGAGGTCGGCACCTTTGGCATGTGGTCCAGCGTGTACGATAACACCCTAAATAATCCTCTCAATAATATCAGCAAAGCAGGCGGCCCCATTGATACCTTCGATGATTATGATTTAGATACTCAGCTCCAATGGCTTGATACCAACGACAACAATAACGTGACGATCCGTGCCGCATGGGTAAACGAGCAGCAGCAATTTGGAGCGGGGAATATCATATCTTCGAACTCCTCCGGTAACTTAAATTTCTTCAATGTTAACGCCACCTACTGGTATCATGACCACTACGGCATTCAGGGCGGATACCGGAATGTGTGGGGGTCCGCTAACCCCGGTCTCTACACTACCACATACACTAATAGTGGTTCTCCAGATACCAGCAATGAATGGATAGAGGCTTCCTATCTGCCGTGGTGGAATACCCGCTTCTCCTTGCGATATGTCGTATACAACAAGTTCAATGGCGTTGGTTCGGCGTCGTCCAACAACCTTGGATATGGGGCGTCTGCGTATAACACCCTTGAACTGCTGGCCTGGATATCATACTAGGAGCCGATGCCATGACGACATACTTAAGCCAAGACCGGTTGCGCAATAAAGAGAACGACACGATGACCTATCAACATAGCAAGATGTATCAGTCGAGAACCTTCCTTCTGTTCAGCGCACTCTTGCTGGTGGCCGGGCAGGCGAGTGCTGCAGTCGGCAGCGCCGACGCGCCGGCACCATACCGCGTCTCCAGTGATTGCATGGTATGCCACGGGATGACGGGCCGTGACACGCTCTATCCGATCGTCCCCCGCCTGGCCGGACAGCATAAGAGTTATATGGAAGCGCAGTTGAAAGCGTATAAGGATCACTCGCGTGCGGATCAGAATGGCGAGATCTACATGTGGCCCGTGGCGCAAGCGCTGGACAGTGCGAAAATCACGGCGCTGGCAGATTACTTCAACGCCCAGAAGCCGCCGATGCAAAGCAGCGGCATCAAGCATGCCGGTGCGAAAGAAGGAAAGGCCATATTCAACCAAGGGGTTACCAACGAACAAATCCCTGCCTGTATGGAATGCCACGGATCGGATGGCCAAGGGGCGGGCCCGTTCCCCCGGCTGGCGGGCCAGCGTTACGGCTACATCATTCAGCAGTTGACCTACTTCCACAACGGCACACGGGTAAATACCCTGATGAACCAGATTGCGAAGAATATCACCGTGGCGCAGATGAAGGATGTGGCGGCTTATCTTTCATCGCTGTAAGCGTTGTAATTGGTCAATAGAAGTTTTCCTGGCAGGCTGAAGTTTATAAAAATGGGTCTGCCAGGCATTTGCACCGTCAGGTTTATGTGCTTCTCAAAGGAGGTAGAGGTATGGCAGCAAAAAAAGGTATGACTACGGTGCTTGTATCCGCCGTGATATGCGCGGGGGTAATTATAGGTGCCCTGGAGTGGGAAAAAGCGGTAGCCCTGCCCAATCCTTCCGGGCAGGTCATTAATGGGGTACATCATTATACGATCGATGAGTTCAACTATTATTATAAACCGGATCGCATGACCTGGCATGTCGGGGAAAAAGTGGAGTTGACGATTGATAACCGATCGCAATCAGCGCCCCCGATTGCGCATCAGTTCTCCATCGGCAGAACGCTGGTATCCCGGGACAATGGCTTTCCAAAATCACAGGCTATCGCCGTGGGATGGAAAGATAACTTCTTTGATGGTGTGCCGATTACCAGCGGGGGACAGACAGGGCCAGTACCGGCGTTTTCCGTCAGCCTCAACGGTGGACAAAAGTACACCTTCAGTTTTGTGGTGCCCAATAAGCCCGGAAAATGGGAATATGGGTGTTTTCTGCAGACGGGTCAACACTTCATGAATGGGATGCATGGTATTCTTGACATACTACCTGCTCAGGGAAGCTAATTTAGGGAGGGCATATGAACGCAGCAAAAGAAAACTTATGGAAAGCTTTCCGCGGCTTGGTGGTGGTCTGGATTATTGGCCTGGCGATTTTCGAAACGCTGATGGCCTGGGGTATCGGTAACTGGCCAATTTTGGGGAGTATTCAGGCGCATATTACCGCAGATGCCACCACATACCTGTTGTGGCAGGCCGTATTCATCTATGTGCTGGTCGGCGGTGCGATTGTATATAGCGCATTTCGTTTCCGCGCATCATCCATGTCAGACACCGCGGCGCCGGCTTATCAAAAACGGACCTGGGCGCCTTTCGTGGTGACCTGGCTGGTTTTGGCCATAGGCATCAACCTGGCAAATACCATTTATCCGGGTATGGTGGGTCTGGAACAACTTTGGGGTATCCAGTTAGATACGAAGAACCCATTGGTGATCGATGTTACCGCGCAACAGTGGAAGTGGACGTTCTCTTATCCTAAGCAGGGCGTAACGGATGTGTCACAACTGGTGGTTCCCGAGGGCCGCACCATATACTTCGTTCTGCGGACAAAGGATGTCATGCACGATTTTTGGGTGCCTGCCTGGGGTGAGAAAAAAGATGTGATCCCCAATGAAGTGCGGCACTTGTTTATTACACCCACCATGTTGGGGACAACCGCTACAAACCCCATGCTGCGTGTACAGTGTTCCTTGATTTGTGGCAACGGACATCCGTTGATGCGCGCTCCGGTGAAAGTGGTAACGCCAGCGGACTTCAAGGCTTGGGTGGCAAACAATAGCTTCTAGTAAAGCCAACGGAAGGCTTGCCAGCACCCAACGTTAAATGTACTAAGGAGTAAGTAATGGCAACTAACGAAATTCAGGAAAATGCGTTGAACAATACGGGAGTGGACAAGACCCCATTTGCGGCTAGCATGCTGTTTCCGCTGTTCCGTGCGACGCTTTGGGGACTAACCGGCTATTTTGCTGCGGCATGGATCACTGCTTTATTGCTCCACACGGTAATCGTAAACCCTTTACCCGCGACAGTGGGTTATGTGGCCGGCTTGGTCTGCTGGCTGATGGGCAGCGGTGTATGGGAGGGATGGATACGACGCGCATTTGGAGGAAAAGAAGCTCCAACTTACACGGGTATCGAACGTTATTTTCGCTTTGGTCCCGATTCAAAATCCGCAGCCGTACGCTACGTAATCTTAAATATACTAACGTTCTGCTTTGCCGGCATGGCCGCCATGGCGATCCGCATTGAACTGTTGACGCCAGACTCCACCAGTTGGTGGCTGTCAGAAATCCAGTACAACCAAACGTTCGGTATTCATGGATTGATGATGTTGTTGGGTGTGGTGGCCTCTGCCATCGTCGGCGGTGTTGGCTACTATCTTATCCCGTTGATGCTTGGCACGAGAAATGTAGTATTCCCAAAACTTCTTGGCCTAAGTTGGTGGCTTTTGCCACCGGCGACCTTCGCTGTTTTTATGAGTCCTACGACCGGTGGGTTTCAGACGGGATGGTGGGGATATCCGCCGTTGGCGCAAAACAGTGGTAGCGGTATTGTGTGGTATGTCCTCGGTGCCGCCACCATTCTTGTTGCGTCGCTACTTGGAGCCATCAATATCGCCGGAACCATGGTGTACATGCGCGCCAAGGGCATGAGCCTGGGTCGCGTTCCGATTTTTGTGTGGGGTTTATTTGCGGCAGCCACCACTCTCGTCGTAGAGTCGCCAGCAACCTATACCGGCGCGCTCATGGACTTATCCGACATGATCGCCGGATCGCATTTTTATACCGGTCCCACCGGCCACCCGTTAGCGTATCTCGATCAGTTCTGGTTTTTGTTCCATCCAGAGGTCTACGTTTTCATTCTGCCCGCTTTTGCCATATGGCTGGAGATTCTTCCTGCCGCGGCCAAGCGGCCGTTGTTTGCTAGGGGTTGGGCCATCGCCGGACTGGTTGGCGTTTCCATGTCGGGTGCAATGTCGGGTGTCCATCACTACTTCACTGCGGTGAGTGACGCGCGTATGCCCATATTCATGACCATAACGGAAACTGTATCCATTCCGACAGGGTTCATTTATTTGTCCGCCATCGGAACGATATGGGGTGGTCGTTTAAGAATTAATGCTGCGGTATTGCTCGTACTGATGGCGATGATGAACTTCCTGATCGGTGGGCTGACGGGCATATTCAATGCCGACGTTCCCGCCGACCTTCAGCTGCACAACACCTACTGGGTTATTGCGCATTTCCATATACGATGCTTTGGTGGAGTGATCTTTACCTGGATTGCCGCGCTATACTGGTGGTTTCCCAAGGTTACTGGACGGAAGATCAATGAATTTTGGGGAAAGTTTCACGCATGGTGGTCCTTCGTATTCTTCAATTGTACGTTCTTTCCCATGTTTATAGCTGGACTAGATGGAATGAACAGGAGAATTGCGATATATCTTCCTTACCTGCATGACATCAACCTGTTTATGTCTATTTCATCCTTTTTCTTGGGCGCAGGGTTTCTCATTCCGCTGGCCAATCTTTTATACAGTTGGCGCTATGGGCCAAAGGCCGAAGCTAACCCTTGGGGCAGCAACGGCCTGGAATGGCAAATAAAATCGCCAACACCGTATGTGCCATATCCAGCAGGAACGGAGCCAGAGGTTGTGGGCCCGAACGATAACTACGCGGCGGAAGCAAAAGACCCCTTTATTTGGGTGTCTACGCCCAGCAAGTAAATTAGAAGGAGTTGAACCATGACAGACAACAGTTATGCCAAGCTAATGGATCCGGCCTCGGAGCGTGCAAAAAGGGGTGCGTTCTTTTTCCTGATGCTTTTTGCAGCCATCATTTTTGCGATGTGGGACCTCGCGCGTTTTCTGTGGGGGCACTCGGTGCCCGCTACATTGAGCATGGGCGTGGGTGTTGCGCTGACTGTTCTGATGCTCGTCAGCCTGGTGCCGGTGATGACGGCCCGCAAAAAACTGGATCAGGGCGATGATGCCGGTATCGTGAGCAGTCTGGCAACCCTGATGGTGGTCTCGTTGGTGATGGCGGGTGGAATCGTCTACAACTGGACTACCTTAACCATCGGTAGTGGTTATGGCGGGATTTATGACATCACCAGCTTGTGGTTTCTGGTACATTTCGTGGCGGCCATCCTGGCGCTGCTGGCGAGTATCATGAAAATCACTCGCACTCCAGAGCGCGCGAAACGCGAGCGATGGGTGTCGTATAACGTGTTAACCTTCTGGGGCGGTGTGATTGTTCTATGGGTTGCATTTTTTATTGTTTTCTATATTGCGTAATGCAGTTTAGAAGATTCTCTAATGGAGTGAGGGTTAGATAATGGATATGTCACATTTATCGTTCGTTATCCCGTCTGGAGCTGATGATCCGACGTTTTTCTGGCTGACGGGGTACATTGGGTTTCCTGTGGTGTTTCTGAGTGCATACTTTTGGTGGGTATTAAAGGAGGCAAGCAAGGAAGATCGGCTGCGTATTCTAAAAAAGGGAGAAGACGGCGCATCTGGAAACGCATGATGTTCCACGGATGGTCGTGCGAGTACCGGGCGGCCATCCGGAGTTGTTTTGCGTTTTACTGTTGCGACGTCGTTATCCATGCTTCAAAGGAGGTAAATCATGAACAAGGAAGGCTGTTTAATTTCTCACGATGATCGCGATGATGGCGCATGGGATGGAAACATCGTGTTGATCATAGGATTATTGTGGGCTATTATTGCTCTGGGTGGCTATTATGTTACCCTTAGAGTGCTGTTTTGAGACAATTCCCCGGCTGGATAGGGCGATGAATACCATGTAGTAGCATATTAAAATGCCAGAGGGCCCGGTGATGGTTTTGTAGGGCGGCTGGTTCTACTCAGGTTAAACGTTAAGGAGAAGGGATAACTTATGTATACACAGAACACGATGAAAAAGAACTGGTATGTGACTGTTGGTGCGGCTGCGGCTCTGGCGGCAACGGTCGGCATGGGTACCGCGATGGCCGGCACGCTGGATTCCACATGGAAAGAGGCGACGCTTCCCCAAGTTAAGGCCATGCTGGAGAAAGATACCGGGAAAGTCAGTGGCGATACAGTTACCTACAGCGGCAAGACTGTACATGTGGTCGCGGCGGCCGTGCTCCCGGGATTTCCGTTCCCGAGCTTTGAAGTTCATGACAAAAAGAACCCGACCTTGGAGATTCCCGCAGGGGCAACCGTAGACGTGACCTTCATTAACACCAACAAGGGATTTGGTCATAGTTTTGACATCACTAAAAAAGGACCGCCTTATGCGGTTATGCCGGTGATTGACCCCATTGTCGCAGGAACTGGATTTAGCCCGGTCCCAAAAGACGGCAAGTTCGGATATACGGATTTCACCTGGCATCCGACGGCGGGTACTTACTACTACGTATGTCAGATACCGGGGCATGCCGCCACCGGTATGTTTGGTAAAATCATTGTCAAGTAAGTCCTGGATGGTTGTTGTCTGGGCAGCTGTGCTTTGCTAGTGTAGGTCCTGGTGGCCAGGGCAAATGGTTATCTTGCCCTGGCCATTGGTATTTATTATAAAATACGAATTTCATGTATTGCGTTATGCTTTGTATGATGTTATGAGTATGTTTGCATGCAACATATGATGATTGATCTAGTTTATTAAGCTATGGACCACGAAAACACGCTGCCTCGGTACATATATTAATTCATTCAGATAAAGTCCCAAACTCAGATATCCTGACG
The sequence is over 8 kb long. So, I would suggest the use of cosmids for cloning. The cosmid can be inserted into E. coli, and be cloned. Inside the E. coli, the sequence replicates, transcripts, and finally translates into protein. The protein from this gene is found on the outer membrane of Acidithiobacillus ferrooxidans. But, since signal peptides and chaperone proteins for the desired protein is missing in the sequence, my educated guess is that it will be found intracellularly, and must be extracted and purified for further investigations.
Alternatively, the cell free method PURE (Protein synthesis Using Recombinant Elements) can also be used because of its faster turn around times. The DNA template strand is incubated in the presence of specific enzymes and cell extracts. The protein obtained must be purified through affinity chormatography.
a. What DNA would you want to sequence (e.g., read) and why? I would love to sequence the antifreeze protein gene from Leucosporidium sp..The protein has a lot of applications in food technology, and medicine, and I would love to produce it commercially.
I
b. What technology or technologies would you use to perform sequencing on your DNA and why? I would use SMRT (Single Molecule Real Time) sequencing technology from PacBio. It can generate long reads (10-25 kb) with Q40+ accuracy. It is also best used for de novo genome assembly.
c. Is your method first-, second-, or third-generation (or other)? It is a third generation sequencing method.
d. What is your input? How do you prepare your input (fragmentation, adapter ligation, PCR)? i) DNA has to be extracted and must be purified to make it free from proteins and RNA. Long and unbroken molecules are considered to be ideal. Freshly exracted DNA is preferred over stored one. ii) The DNA is enzymatically sheared into 10-25 kb long fragments iii) SMRTbell library format is preferred for the preparation of library, where hairpin adapters are ligated to both 5’ and 3’ ends to create a circular template of DNA fragment.
iv) Sequencing primers and the appropriate polymerases are added to the buffer containing the DNA.
v) It is then loaded on to SMRT cell, that contain zero-mode waveguides. Each ZMW captures a single DNA molecule for sequencing.
e. List the essential steps. Answered above.
f. How does your chosen sequencing technology decode bases (base calling)? Each nucleotide contains a unique fluorescent labels, which get excited with a laser whenever a new base is added. The instrument then records the color and timing of each flash, which corresponds to the base that has been added.
g. What is the output? HiFi reads, usually 10-25 kb long are obtained as output.
5.2 DNA Write
a. What DNA would you want to synthesize (e.g., write) and why? I want to synthesize the Temporin 1 CE A gene found in frogs. It is a small peptide antimicrobial, and can be used to combat antibiotic-resistant bacteria.
b. What technology or technologies would you use to perform DNA synthesis and why? Phosphoaramidite method, followed by Gibson assembly can be used to synthesize it.
c. Essential steps of chosen synthesis methods See Homeowrk 1 for the steps of phosphoaramidite synthesis. Steps of Gibson Assembly:
Mix the pure, synthesized fragments into the reacton mix containing 5’ exonuclease, DNA polymerase, and DNA ligase. It is essential to ensure that the synthesized fragments have 15-30 bp overalaps to prevent random ligations.
Incubate the samples at 50 degree celcius. This ensures that only cannonical base pairing (A=T and G≡C) occurs and non canonical bonds are prevented due to their instability at this temperature.
exonuclease cleaves the -OH group, polymerase adds the nucleotides, and ligase binds the sequneces together.
d. Limitations (speed, accuracy, scalability) Phosphoaramidite method is limited by its inability to synthesise fragments longer than 200 bp, poor yields for longer fragments, and relatively higher cost per synthesized base pairs. Gibson assembly is limited by its dependency on overlaping fragments that need to be precise. The assembled sequences must be sequenced again to make sure that it is accurate and misjoins and mutations have not occured.
5.3 DNA Edit
a. What DNA would you want to edit and why? I would edit RSL4 gene in plants, since its overexpression increases root hair length. Longer root hairs allow the plant to uptake more nutrients.
b. What technology or technologies would you use to perform DNA edits and why? I would use CRISPR/Cas9 because it allows precise, targeted edits and can be adapted for either gene activation or promoter replacement to drive RSL4 overexpression.
c. How does your technology edit DNA? CRISPR/Cas9 uses a guide RNA to direct the Cas9 nuclease to a specific DNA sequence, where it introduces a double‑strand break. Repair pathways or engineered activators then modify or enhance gene expression.
d. Essential steps
Design guide RNAs targeting the RSL4 promoter or coding region
Clone them into a CRISPR vector
Deliver the construct into plant cells
Select transformed cells and regenerate whole plants
e. Preparation needed (design steps) Identify target sites in the RSL4 promoter, ensure PAM sequences are present, and design guide RNAs with minimal off‑target potential. Choose a strong promoter or CRISPR activation system to boost expression.
f. Inputs (DNA template, enzymes, plasmids, primers, guides, cells) Inputs include the RSL4 gene sequence, Cas9 enzyme, guide RNAs, plasmid vectors with promoters, plant cells for transformation, and primers for verification PCR.
g. Limitations (efficiency, precision) CRISPR editing efficiency can vary across plant species, and off‑target effects may occur. Regeneration of edited plants is time‑consuming, and overexpression may cause unintended growth trade‑offs.
Keywords I would use to describe it: Opentrons OT-2, automation, standradization, synthetic biology.
Summary of the paper: The authors developed an open-source software called ‘Slowpoke’ to automate the Golden Gate assembly process. Opentrons were used to carry out bacterial transformation, GG assembly, and plating. After a few manual steps in between, Opentrons were used once again to perform cPCR. It demonstrated the feasiblity of automating GG assembly.
Opentrons were used to handle liquid transfers, reaction mixtures, and parameters. By integrating pipetting, transformataion, plating, and cPCR screening into a single pipleline. The validation was carried out manually using flow cytometry with transformed yeast cells. Using Slowpoke interface along with Opentrons, the authors achieved high assembly efficiencies, over 90% with Yeast Toolkit (YTK) and 60% with Subtilis Toolkit (STK), consistent with values reported for manual Golden Gate assemblies using these toolkits.
To conclude, this paper designed a tool (Slowpoke) that generates Opentrons-ready protocols in the form of CSV files, mitigating the expertise needed in coding to a great extent. However, it must be noted that human input was still necessary to collect the DNA fragments for running cPCR.
v
2. Write a description about what you intend to do with automation tools for your final project. You may include example pseudocode, Python scripts, 3D printed holders, a plan for how to use Ginkgo Nebula, and more. You may reference this week’s recitation slide deck for lab automation details.
I would like to automate the prototyping of a novel Bio In-situ Resource Utilization (Bio-ISRU) on Mars that comprises of two trophic levels. The producer level utilizes photoautotrophic organisms to convert Martian CO2 (and of course, sunlight) to produce the nutrients required for the primary consumer level. The latter would consist of a ‘biominer’- a bacteria that can precipitate, from the Martian regolith, metals- cheifly iron in the form of Fe3O4- for easier metallurgical applications.
The automation tools would be utilized in the following ways:
3D printing of a photobioreactor in gyroid shape to maximize the surface area for photosynthesis. This has to be validated in Martain gravity. Probably, can be carried out on a space station using a centrifuging apparatus to mimic the higher gravitational pull on Mars compared to Low Earth Orbit (LEO)
Bioreactor management (i.e, addition of nutrient media etc.)
Sensing if the maximum biomass has been achieved, and if yes, lysing the cells so that they may be utilized by the biominers
Efficient mixing of the lysed biomass with the Martian Regolith at appropriate ratio to maximize the precipitation of Fe3O4 by Acidithiobacillus spp.
Sensing the maximum quantity of Fe3O4 precipitaed, and removing it by operating a magnetic arm to separate the magnetite.
Sterilization of equippment, as well as decontamination, using gamma radiation.
Pseudocode (The following was the output of Gemini 3 for the prompt: “Write a pseudocode for the following”, and the above block was pasted.):
import ginkgo_nebula_api as nebula
# Configuration Constants
MARS_GRAVITY_RPM = 24.5 # Calculated RPM for centrifuge to mimic 0.38g
BIOMASS_THRESHOLD = 0.85 # OD600 value for harvest
MINING_RATIO = 0.4 # Ratio of Lysate to Regolith
IRON_SATURATION_POINT = 0.92 # Signal from Magnetic Flux sensor
class BioISRU_System:
def __init__(self):
self.stage = "PRODUCER_GROWTH"
self.centrifuge_active = True
def run_lifecycle(self):
# 1. GRAVITY VALIDATION
# Maintain Mars-simulated gravity via centrifugation
if self.centrifuge_active:
mars.centrifuge.set_speed(MARS_GRAVITY_RPM)
# 2. PRODUCER STAGE (Cyanobacteria)
if self.stage == "PRODUCER_GROWTH":
mars.pbr.inject_media(interval="6h")
current_od = mars.pbr.read_sensor("OD600")
# Check if gyroid surface area is maximized with biomass
if current_od >= BIOMASS_THRESHOLD:
print("Maximum biomass achieved. Initiating Lysing...")
self.stage = "LYSING"
# 3. LYSING & HANDOVER
if self.stage == "LYSING":
mars.pbr.activate_ultrasonic_lyser(duration="10m")
# Transfer to the Miner Tank
lysate_volume = mars.pbr.get_volume()
mars.transfer_pump.move(to="MINER_TANK", volume=lysate_volume)
self.stage = "BIO_MINING"
# 4. CONSUMER STAGE (Biominers + Regolith)
if self.stage == "BIO_MINING":
# Automated mixing of lysed biomass and Martian Regolith
regolith_amount = mars.transfer_pump.last_volume * MINING_RATIO
mars.regolith_feeder.add(regolith_amount)
# Efficient mixing to maximize Acidithiobacillus contact
mars.miner_tank.start_agitation(pattern="vortex", speed="medium")
# 5. SENSING & MAGNETITE HARVEST
iron_yield = mars.miner_tank.read_sensor("MAGNETIC_FLUX")
if iron_yield >= IRON_SATURATION_POINT:
print("Magnetite precipitation peaked. Deploying Magnetic Arm.")
mars.magnetic_arm.extend()
mars.magnetic_arm.energize()
mars.magnetic_arm.extract_to(bin="IRON_STORAGE")
self.stage = "DECONTAMINATION"
# 6. STERILIZATION & RESET
if self.stage == "DECONTAMINATION":
print("Initiating Gamma Radiation Sterilization cycle...")
mars.gamma_source.expose(duration="30m", target="ALL_CHAMBERS")
# Uplink yield data to Ginkgo Nebula for strain optimization
nebula.upload_log(yield_data=iron_yield, efficiency=1.2)
# Reset for next cycle
self.stage = "PRODUCER_GROWTH"
print("System Reset. Starting new ISRU cycle.")
# Initialize and Loop
isru_unit = BioISRU_System()
while True:
isru_unit.run_lifecycle()```
### Final Project Ideas:
[Google Slides](https://docs.google.com/presentation/d/1FAFN4YYisOcso3CI5F3W3Z7hj6_n9D1vAhVUywQXKPU/edit?slide=id.g3ca9627a0a6_624_27#slide=id.g3ca9627a0a6_624_27)
