Tool Description Biosensor for the detection of stress or diseases in plants useing Escherichia coli chassis.
In this system, stress signals or specific markers associated with certain pathogens induce the production of a fluorescent protein like GFP. This biosensor could be used as a tool for the early detection of plant pathologies by exposing the bacteria to the plant extracts or exudates. T
Part 1: Benchling & In-silico Gel Art I design two gels:
Gel A: ‘ART’
Gel B: ‘ATP’
Part 3: DNA Design Challenge 3.1. Choose a protein - TcHMGB For this homework, I chose the protein HMGB from Trypanosoma cruzi (TcHMGB), the parasite that causes Chagas disease. TcHMGB was characterized by the research group in which I carried out my undergraduate thesis. During this work, I constructed a DM28c cell line expressing TcHMGB fused to the biotin ligase TurboID, with the objective of performing a proximity labeling–based interactomics assay.
Biosensor for the detection of stress or diseases in plants useing Escherichia coli chassis.
In this system, stress signals or specific markers associated with certain pathogens induce the production of a fluorescent protein like GFP. This biosensor could be used as a tool for the early detection of plant pathologies by exposing the bacteria to the plant extracts or exudates. T
he system could be build:
a. to respond to a group of markers related to plant infection or to a group of markers for a certain pathogen.
b. to detect if the plant is under abiotic stress or is infected, resulting in the production of different signals depending on the diaggnose.
Plants under abiotic stress and plants affected by pathogens often show similar external symptoms, but at a molecular level these two conditions can be differentiated. A biosensor capable of responding differentially to this conditions or only to one of these conditions could help distinguish between them, enabling more appropiate interventions
Governance Goals
Ambiental Security: we must avoid ambiental damage
Use a non pathogen strain as a chasis
the biosensor must be manipulated in a laboratory
propeer disposal of the material that has been in contact with the biosensor
Proper use of the tool
Inclusion
disminish costs
make the tool accesible for small productors
Responsible interpretation and use of results: missinterpretation of the output could lead to incorrect interventions
Ensure that the biosentor results are presented as screening tools
Provide clear guidance on the limitantions of the biosinsor´s accuracy
Governance Actions
Stablish requirements of use
Purpose:The biosensor is based on a genetically modified organism. This action proposes establishing its use exclusively in laboratories with the required biosafety level in order to avoid the accidental spread of the organism into the ecosystem.
Design: For this to work, a group of professionals must create a guide for use and establish requirements for purchasing the product. Users of the product must follow the provided guidelines and instructions.
Assumptions: It is assumed that trained personnel will follow the guide for use and will not misuse the product.
Risks of Failure & “Success”: Unresponsible personnel may misuse the biosensor, leading to the accidental release of the bacteria into the environment.
Propper disposal of the material
Purpose: Ensure that users have the appropriate means for the proper disposal of materials, and monitor correct disposal in order to prevent environmental contamination.
Design: Institutions and laboratories using the iosensor must provide approved disposal systems for biological waste, such as sterilization or inactivation procedures. Clear disposal protocols must be included in the user guide, and compliance should be overseen by institutional biosafety committees.
Assumptions: It is assumed that that users will follow established disposal protocols.
Risks of Failure & “Success”: disposal procedures may not be properly followed or enforced, leading to unintended release of genetically modified bacteria.
Result interpretation guidelines
Purpose:Prevent misinterpretation of biosensor outputs by ensuring that results are understood as indicative signals rather than definitive diagnoses.
Design:Develop standardized interpretation guidelines that accompany the biosensor, including clear explanations of what a positive or negative signal means and its limitations. These guidelines should be created by academic experts and included in the user manual.
Assumptions: It is assumed that users follow the interpretation guidelines when analyzing results.
Risks of Failure & “Success”: users may ignore or oversimplify the guidelines.
Does the option:
Requierments of use
Proper desposal
Result interpretation
Enhance Biosecurity
• By preventing incidents
1
2
n/a
• By helping respond
2
2
n/a
Foster Lab Safety
• By preventing incident
1
1
3
• By helping respond
2
2
3
Protect the environment
• By preventing incidents
1
1
n/a
• By helping respond
2
2
n/a
Other considerations
• Minimizing costs and burdens to stakeholders
3
2
1
• Feasibility?
2
1
1
• Not impede research
2
2
1
• Promote constructive applications
2
2
1
Priorization of gocernance options
Based on the scoring, the governance options that should be prioritized are establishing requirements of use and result interpretation guidelines, while proper disposal of materials functions as a complementary measure.
Requirements of use are essential because they directly reduce risks related to biosecurity, laboratory safety, and environmental protection by limiting the biosensor to controlled laboratory settings. However, these requirements may increase costs and restrict access, particularly for smaller laboratories.
Result interpretation guidelines are a high-priority complementary action because they are easy to implement, low cost, and help prevent incorrect decisions based on biosensor outputs. Since the biosensor is intended as a screening tool, clear guidance is necessary to avoid inappropriate interventions.
Proper disposal of materials is also important but relies strongly on infrastructure and enforcement, which can vary between institutions.
Homework Questions from Professor Jacobson:
Nature’s machinery for copying DNA is called polymerase.
What is the error rate of polymerase? 1/106
How does this compare to the length of the human genome? The human genome is approximately 3 × 10⁹ base pairs long. At this error rate, thousands of errors would be expected per replication cycle if no correction mechanisms were present
How does biology deal with that discrepancy?1. polymerases can detect and corect errors (proofreading activity); 2. Cells have repear sytems to correct DNA mutations (like MutS system)
How many different ways are there to code (DNA nucleotide code) for an average human protein? There are many ways to code for an average human protein due to the degeneracy of the genetic code: each aminocid is encoded by two or three codons.
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 use of codons differs between organisms; that is, some organisms commonly use codons that are rare in others. As a consequence, the translation efficiency of a human protein expressed in yeast or bacteria can be limited due to the low availability of the corresponding tRNAs. Another reason is the absence or differences in post-transcriptional and post-translational modifications between expression systems (such as bacteria or yeast) and human cells, which can affect protein folding, stability, and functionality.
Homework Questions from Dr. LeProust:
What’s the most commonly used method for oligo synthesis currently? Solid-phase chemical synthesis, specifically phosphoramidite chemistry, is the most widely used method for oligonucleotide synthesis.
Why is it difficult to make oligos longer than 200nt via direct synthesis? Because error in nucleotid addition accumulates with increasing length, leading to a sharp drop in yield and sequence fidelity for longer oligonucleotides.
Why can’t you make a 2000bp gene via direct oligo synthesis? Considerieng the avobe, direct syhthesis of a 2000bp gene would result in an unacceptably high error rate and extremely low yield.
Homework Question from George Church:
[Using Google & Prof. Church’s slide #4]
What are the 10 essential amino acids in all animals? **H, I, L K, M, F(Phe), T, W, V and R.
How does this affect your view of the “Lysine Contingency”? The “Lysine Contigency” could have made scence if the dinosaurs had have been genetically engineered to be able to produce all essential amino acids except lysine, so that they would survive only with lysine supplementation (and not the rest of the essential amino acids), and die when supplementation was withdrawn.
IA prompts:
Q: Which are the 10 essential aa in animals?
A:The 10 essential amino acids in animals are:
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Arginine
Week 2 HW: DNA Read, Write, & Edit
Part 1: Benchling & In-silico Gel Art
I design two gels:
Gel A: ‘ART’
Gel B: ‘ATP’
Part 3: DNA Design Challenge
3.1. Choose a protein - TcHMGB
For this homework, I chose the protein HMGB from Trypanosoma cruzi (TcHMGB), the parasite that causes Chagas disease. TcHMGB was characterized by the research group in which I carried out my undergraduate thesis. During this work, I constructed a DM28c cell line expressing TcHMGB fused to the biotin ligase TurboID, with the objective of performing a proximity labeling–based interactomics assay.
TcHMGB belongs to the HMG-B family, which plays a key role in chromatin organization and gene regulation in eukaryotic organisms. TcHMGB has been shown to modify chromatin structure, making it more accessible to the replication, transcription, repair, and recombination machinery. Additionally, TcHMGB is believed to be essential for T. cruzi survival and is therefore considered a potential therapeutic target.
Protein sequence
Found in UniProt
tr|Q4D714|Q4D714_TRYCC High mobility group protein, putative OS=Trypanosoma cruzi (strain CL Brener) OX=353153 GN=Tc00.1047053504431.64 PE=4 SV=1
Codon usage differs between organisms; that is, some organisms preferentially use codons that are rare in others. As a consequence, the translation efficiency of a T. cruzi gene expressed in yeast may be limited by the low availability of the corresponding tRNAs. Therefore, codon optimization is required to improve the efficiency of expression of the protein of interest.
tchmgb encodes a protein from Trypanosoma cruzi, a unicellular eukaryotic organism. Therefore, Saccharomyces cerevisiae was selected as the expression host because it provides an eukaryotic cellular context, including post-translational modification and protein-folding machinery, which is absent in prokaryotic systems such as Escherichia coli.
tcHMGB was codon-optimized for expression in Saccharomyces cerevisiae using the IDT Codon Optimization Tool (Integrated DNA Technologies; https://www.idtdna.com/CodonOpt)
ATG TCA ACT GAA CTG AAG AGT GGC CCA TTG CCT GCA GAC GTA GAA GAG GTC ATA GCT AAT ATT ATG AGG GAA GAA GGA GTG AAT TTT TTG ACT TCT AAG ATT CTT AGA CTT AGA CTA GAA GCA AGA TAC AGA ATG GAA TTT ACT TCA CAT AAA GCC GCT ATT GAA GGC ATC ATT ACA AAA TTA ATG CAG TTG CCT GAA TTT AAG AAG CAA TTA GAG AAC GCA GTA AAA GAA GAG AAG GCA GCG TCA TCT ATT GGA GGT AAA AAG AAA AAG AGA TCA GCT TCT GCG GCT GCA GAC GAA AGA AAT GCT AAG GTC AAC AAG AAG GAA AAA AAA CCT GAC GAT TAT CCA AAG GCA GCT TTG AGC CCA TAT ATT TTA TTC GGT AAT GAC CAT AGA GAT AAG GTG AAG GAA CAA AAT CCA GAA ATG AAA AAT ACT GAG ATT TTG CAA TCA TTG GGA AAA ATG TGG GCC GAG GCG TCT GAC GCA GTT AAA GAG AAA TAC AAG AAA TTA GCC GAA GAT GAT AAG AAA CGT TTC GAT AGA GAA TTA TCT GAA TAC AAA AAA AGT GGT GGT ACT GAA TAT AAA CGT GGA GGT GGC AAG GTT AAA GCT AAG GAT GAG AAC GCT CCT AAG CGT AGT ATG TCT GCA TAT TTT TTT TTC GTG AGT GAT TTC AGA AAA AAG CAT CCA GAC TTA AGT GTT ACA GAA ACC TCT AAA GCC GCC GGT GCT GCT TGG AAA GAA CTA TCC GAT GAG ATG AAA AAG CCA TAC GAA GCT ATG GCA CAG AAG GAT AAA GAA AGA TAT CAA AGA GAA ATG GCT GCT AGG GCG
3.4. You have a sequence! Now what?
To produce this protein, it is first necessary to obtain a plasmid that enables expression of the protein of interest in the selected expression system, either cell-dependent or cell-free. For plasmid construction, there are two main options: (1) cloning the coding sequence into an existing expression vector, or (2) ordering a synthetic plasmid that includes regulatory elements selected according to the chosen expression system and experimental goals—such as a constitutive or inducible promoter, ribosome binding site, coding sequence, affinity tag, and transcription termination sequence—thereby improving expression efficiency and control.
Once the plasmid is obtained, it is introduced into the chosen expression system. In this case, Saccharomyces cerevisiae was selected as the host organism. The plasmid can be delivered into yeast cells by electroporation, followed by selection of successfully transformed cells. After selection, protein expression is induced under the appropriate conditions, allowing transcription of the gene and subsequent translation into the protein of interest.
Since I could not use Twist directly for this exercise, I downloaded the pTwist Amp High Copy cloning vector map and imported it into Benchling. I then inserted the previously designed TcHMGB expression cassette into this backbone using Benchling’s sequence editing tools, allowing visualization and validation of the complete circular plasmid.
Part 5: DNA Read/Write/Edit
5.1 DNA Read
(i) What DNA would you want to sequence (e.g., read) and why?
I would choose to sequence DNA from hospital wastewater.Hospital effluents contain a high diversity of bacteria exposed to strong antibiotic selective pressure, making them important reservoirs of antimicrobial resistance genes. By sequencing this DNA, it is possible to identify and monitor resistance genes and their potential spread into the environment.
This information is highly relevant for public health, as it can contribute to early detection of emerging resistance patterns and support strategies to limit the dissemination of antibiotic resistance.
(ii) In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why?
I would use Oxford Nanopore to obtain long reads that provide structural and genetic context, and Illumina to obtain short, high-quality reads that allow more reliable genome assembly and error correction.
Is your method first-, second- or third-generation or other? How so?
Illumina is a second-generation sequencing technology, as it relies on sequencing-by-synthesis and generates large numbers of short, highly accurate reads in parallel. Oxford Nanopore is considered a third-generation sequencing technology because it sequences single DNA molecules directly and produces long reads without requiring amplificatio.
What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.
My input would be total DNA extracted from hospital wastewater, which includes mixed microbial DNA from a metagenomic sample.
Essentially, Illumina requires DNA fragmentation and amplification, while Oxford Nanopore can sequence long, minimally processed DNA.
The essential steps are:
High-quality DNA extraction
Library preparation, including fragmentation (not necessary for Nanopore), amplification, and incorporation of adapters and indices
3.Sequencing
What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?
Illumina decodes DNA by detecting fluorescent signals emitted when labeled nucleotides are incorporated during DNA synthesis. Each base is identified based on its specific fluorescence signal.
Oxford Nanopore decodes DNA by measuring changes in ionic current as a DNA strand passes through a nanopore. Different nucleotide sequences cause characteristic current disruptions, which are translated into base calls using computational algorithm.
What is the output of your chosen sequencing technology?
Illumina sequencing results in short, high-quality sequencing reads, while Oxford Nanopore produces long reads of variable length.
In both sequencing technologies, the output consists of FASTQ files containing all the reads, which can be processed using multiple platforms for downstream analysis
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!
(ii) What technology or technologies would you use to perform this DNA synthesis and why?
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?
5.3 DNA Edit
(i) What DNA would you want to edit and why? In class, George shared a variety of ways to edit the genes and genomes of humans and other organisms. Such DNA editing technologies have profound implications for human health, development, and even human longevity and human augmentation. DNA editing is also already commonly leveraged for flora and fauna, for example in nature conservation efforts, (animal/plant restoration, de-extinction), or in agriculture (e.g. plant breeding, nitrogen fixation). What kinds of edits might you want to make to DNA (e.g., human genomes and beyond) and why?
(ii) What technology or technologies would you use to perform these DNA edits and why?
How does your technology of choice edit DNA? What are the essential steps?
What preparation do you need to do (e.g. design steps) and what is the input (e.g. DNA template, enzymes, plasmids, primers, guides, cells) for the editing?
What are the limitations of your editing methods (if any) in terms of efficiency or precision?
Week 3 HW: Lab automatation
Python Script for Opentrons Artwork
To create the Python file to run on an Opentrons liquid-handling robot, I used the scripts from the file downloaded from http://opentrons-art.rcdonovan.com/
as guide, to write the scripts unthe the ‘#YOUR CODE HERE’ line. I also edited the ‘well_color’ library to add some colors.
To create the Python file to run on an Opentrons liquid-handling robot, I used the scripts from the file downloaded from http://opentrons-art.rcdonovan.com/
as guide, to write the scripts unthe the ‘#YOUR CODE HERE’ line. I also edited the ‘well_color’ library to add some colors.
