Individual Final Project
Subsections of Individual Final Project
Project Break Down
DRAFT ABSTRACT:
Air pollution is a pervasive oxidative stressor that disproportionately impacts marginalised urban populations, creating a silent crisis of environmental inequality. Current sensing infrastructure often lacks public visibility, leaving the physical toll of poor air quality abstract and unactionable for the communities most affected.
This conceptual project, ALVEOLI envisions a public art installation designed to create awareness and social engagement, addressing the short falls of how environmental data is communicated and experienced.
I aim to develop 3 bio-hybrid sculptures installed in 3 cities globally, translating invisible atmospheric pollutants into a high-contrast visual readout using engineered microbial biosensors.
Original Project Proposal Slide:
Final Project Slide by Isobel Leonard ---DRAFT AIMS:
Aim 1: Optimise and validate an E. coli extract-based cell-free protein synthesis (CFPS) system compatible with σ⁷⁰ bacterial promoters.
Optimise and validate an E. coli extract-based cell-free protein synthesis (CFPS) system using a constitutive σ⁷⁰ promoter–GFP reporter construct to confirm reliable transcription–translation performance and compatibility with downstream biosensor circuitry in a lyophilised CFPS format. Characterise expression kinetics, signal stability and reproducibility across extract batches to establish baseline system performance for zinc-responsive biosensor development.
Aim 2: Design and validate a zinc particulate responsive CFPS biosensor with colorimetric output
Design a zinc particulate-responsive biosensor based on the ZntR/PzntA regulatory system coupled to a colorimetric reporter compatible with E. coli extract-based CFPS.
Construct DNA sequences in Benchling and prepare constructs for synthesis via Twist Bioscience to confirm manufacturability and robustness of the genetic design.
Develop a lyophilisation protocol and test biosensor response under controlled laboratory conditions, characterising detection limits, signal stability, and colorimetric intensity.
Develop and evaluate a particulate capture, ion-release, and sensor activation workflow enabling conversion of PM2.5-associated zinc into detectable Zn²⁺ prior to CFPS activation, with consideration of the aesthetic and practical constraints of integration into a public artwork.
Aim 3: Integrate the zinc biosensor into a deployable sculptural sensing platform and evaluate performance under environmental conditions
Integrate the CFPS zinc biosensor into a 3D-printed sculptural sensing platform incorporating particulate capture, ion release and lyophilised reaction modules.
Evaluate biosensor performance under environmental conditions, including detection limits, response robustness and signal stability relative to environmental air sample processed in lab conditions.
Use experimental data to guide iterative optimisation strategies such as transcriptional signal amplification and the design of specific riboswitches to improve sensitivity to environmentally bioavailable concentrations of zinc particulate matter.
Finalise a comprehensive public installation proposal and prototype for bio-sensing sculptures in different cities, detailing sculpture placement, maintenance and bio-safety protocols and demonstrating the feasibility of integrating scientific bio-sensing with public art to make air pollution inequality visible and socially engaging.
Apply to funding bodies to pursue realisation of a global project.
Project Breakdown
AIM 1: Establish and validate a σ⁷⁰compatible E. coli extract-based CFPS system
Step 1 : Identify a suitable air pollution biomarker
Evaluate candidate pollutants based on:
- environmental relevance (urban prevalence, toxicity, manmade)
- expected environmental concentration ranges
- compatibility with transcription-factor sensing systems
Output: justification of final selection
Step 2 : Assess bio-sensing compatibility with CFPS
Determine:
- whether known transcription-factor biosensors exist
- which RNA polymerase recognise promoters
- whether sensing requires cofactors absent from CFPS
- whether the pollutant must be chemically converted before detection
- whether the analyte is stable after particulate capture
Output: selected sensing strategy compatible with CFPS transcription machinery
Step 3 : Define CFPS platform requirements
Establish:
- extract type required
- promoter compatibility constraints
- expected signal strength needed for environmental detection
Design validation experiment using a simple reporter construct to confirm:
- transcription reliability
- translation efficiency
- stability after freeze-drying
Output: validate reliable CFPS system
AIM 2: Design and validate a particulate-responsive CFPS biosensor
Step 4 : Select sensing cassette
Select based on:
- specificity
- compatibility with CFPS
- Sequence availability
Output: select sensing mechanism
Step 5 : Select reporting strategy
Evaluate based on:
- visibility in natural lighting
- compatibility with CFPS
- response speed
- signal amplification potential
- stability over time
- compatibility with field deployment
Output: selected reporting cassette
Step 6 : Design genetic circuit logic
Determine
- promoter strength requirements
- RBS compatibility
- appropriate backbone (copy number, origin)
- Strong terminators
- Spacing
and draw out circuit logic.
Validate circuit design in silico prior to Twist order:
In Benchling:
- annotate promoter regions
- confirm ORF integrity
- Insert orientation
- verify reading frame continuity
- prepare synthesis-ready construct
Submit sequence to Twist Bioscience
Output: synthesis-ready circuit design
Step 7: Validate biosensor performance in CFPS
Test under controlled conditions:
Measure:
- response dynamic range
- detection threshold
- signal stability
- response time
- background expression
Evaluate compatibility with freeze-drying workflow.
Output: functional biosensor prototype
Step 8: Develop particulate capture and ion-release workflow
Design strategy for converting particulate-associated pollutants into detectable molecular form.
Evaluate:
- particulate capture system
- ion extraction chemistry compatibility with CFPS
- buffering requirements
- reaction activation
- deployment practicality
- safety considerations
- aesthetics
Output: validated sample preparation workflow
AIM 3: Integrate biosensor into deployable sculptural sensing platform
Step 9 : Integrate biosensor into sculptural structure
Design integration strategy considering:
- placement
- reagent storage
- environmental protection
- replacement accessibility
- temperature stability
- biosafety containment
- maintenance
Output: deployable sensing sculpture prototype
Step 10 : Evaluate environmental performance
Test system under real-world conditions:
Measure:
- detection reliability
- signal visibility
- environmental stability
- variation across sampling locations
- agreement with environmental monitoring datasets and control air sample
Output: field validation dataset
Step 12 : Identify optimisation pathways
Use results to refine:
- sensing sensitivity (riboswitches)
- signal amplification strategy
- environmental robustness
- deployment workflow
- public interface
Output: second spiral biosensor design strategy
Step 13 : Finalise a complete design proposal and prototype
- Finalise a refined design proposal and working prototype for submission to funding bodies for project realisation.
DNA Design
Final Project Slide by Isobel LeonardDNA Design Goal:
A biosensor for the simple, colour change based detection of salicylate and naphthalene in the environmental air.
Based on the methodology of Cho et al (2014) and Park et al (2005), using the NahR/Pr/Psal lower operon from Pseudomonas putida’s Napthalene degrading plasmid NH7 and connecting it to the LacZ reporter gene that produces red-ß-D-galactopyranoside, hydrolysed in CPRG to provide a clear visible signal. The operon uses a constitutive promoter Pr and inducible promoter Psal in opposite directions.
I am trying to create a similarly functioning plasmid in Benchling for an in Silico Aim 1 using the digest and ligation assembly tool.
Reference Paper for Methodology on plasmid construction:
Reference paper for Nah/Pr/Psal Operon:
Plasmid diagram from Cho et al 2014:
In my attempts to create a similar functioning plasmid in silico I have followed the following workflow and then met some problems!
Circuit Logic:
Biobits I need:
Backbone
pGL3b basic (2.9kb) digested and linearised with SaII and HindII and Luciferase reporter gene removed.
Reporter Gene:
LacZ fragment from psV-beta-Galactosidase. (3.7 kb)
Cut out with restriction enzymes SaII and HindIII and ligated into pGL3b basic to make plasmid PGLacZ.
Sensing NahR operon from Pseudomonas putida (1.3 kb)
Containing nahR/Pr/Psal and I have found variants on the sequence located in these places:
Design and add SacI and Xhol cut sites and ligated into plasmid PGLacZ.
Workflow:
Step 1:
- Import pGL3- basic into benchling
- Digest with SalI and HindIII
- Remove luciferase and linearise maintaining origin of replication and antibiotic resistant gene.
Benchling link: https://benchling.com/s/seq-kf6LTrbodved1t066n13?m=slm-UxQtbtTBRIF5SaInZm2z
Step 2:
- Import psV-beta-Galactosidase
- Cut out LacZ fragment with restriction enzymes SalI and HindIII
Benchling link: https://benchling.com/s/seq-R5yEkXzifvA87rugfx2z?m=slm-7vqIszJaDNRN5P8LbkGW
Step 3:
- Ligate intermediary plasmid using Assembly wizard
- select pGL3-Basic as backbone and lacZ as insert
- Ligate to form pGLacZ
Benchling link: https://benchling.com/s/seq-VPPhB7LPfBWE65dQCNoa?m=slm-IZFORq41ceeD4uLW8Cra
Step 4:
- Locate sequence for NahR/Pr/Psal from naphthalene degrading gene cluster from P. putida and annotate.
Versions of sequence I have found:
GenBank: AY294313
- Add restriction binding sites for SacI and Xhol
- Insert upstream of LacZ
Here is where I have hit my problems
Problem 1: Unannotated sequence fragments
As there are no annotated and document sequences for each bit of the NahR/Pr/Psal operon e,g NahR gene, Pr Promoter, Psal promoter and the binding sites used, I don’t know which bit of this gene cluster from GenBank AY294313 is the bit I need.
In addition, I don’t know how to check all the bits I need for a functional sensor e.g RBS, Pr promoter, Psal Promoter are all included in the sequence (which they should be) and if they are the right way round and how to annotate them! I’ve been doing my head in a little trying to work it out!
In the Genbank info it says Gene NahR is 305-1207 and the next gene nahG starts at 1363. Meaning the promoters and binding sites must be between 1207 and 1363 but I am unsure how to identify?
I have tried my best to identify parts of the sequence in this benchling file by cross referencing between different versions of the Nah/Pr/Psal sequence across GenBank and iGEM but I don’t think I’m doing well and if this is a good approach:
These two iGEM entries give a bit more info but are different from the GenBank sequence and I still don’t know how to tell where everything is??
I have the same problem with the LacZ I digested from psV-beta-Galactosidase as although I can see the start and stop codon for the gene, I can’t tell if there is a RBS at the start of the sequence and which one it is?
I don’t know if the problem is that I am taking pieces from different vectors and instead I should try and build the sequence blocks myself with e.g Promoter, RBS, start codon, CDS etc. However, I still have the problem that I am unable to find the sequence for NahR, Pr or Psal promoter within the gene clusters and I wouldn’t know what RBS to use for the LacZ gene.
Problem 2: Multiple SacI cut sites
I have noticed (which is not documented in the paper I am following) that I have 2 SacI cut sites in my new plasmid PGLacZ, because the LacZ I have taken and ligated in has another SacI cut site in it. Meaning that if I were to digest at Xhol and SacI to insert the NahR/Pr/Psal region as described I would get another cut in LacZ.
However, I reason that I can just digest and ligate the pieces in a different order. First digest at Xhol and SacI while the LacZ is not in the backbone. Then digest at HindIII and SalI to remove luciferase and ligate in LacZ.
Is this okay to do??? Or is it better to use Gibson Assembly?? If so how do I design the overlaps for NahR/Pr/Psal so it goes specifically where it needs to go upstream of LacZ.
Problem 3: Cell Free System
I am looking into the possibility of innovating with the biosensor by creating a protocol to use the plasmids in a cell free system to create a safer and more stable bio-sensing public sculptures.
I am interested in merging the research I have already done with the research we learnt about in Cell free systems week from these two papers:
- Ho, G., Kubušová, V., Irabien, C., Li, V., Weinstein, A., Chawla, S., Yeung, D., Mershin, A., Zolotovsky, K., & Mogas-Soldevila, L. (2023). Multiscale design of cell-free biologically active architectural structures. Frontiers in Bioengineering and Biotechnology, 11, 1125156. https://doi.org/10.3389/fbioe.2023.1125156
Where the possibility of cell free biosensors being 3D printed ina biopolymer matrix for architectural structures is discussed.
- Nguyen, P. Q., Soenksen, L. R., Donghia, N. M., Angenent-Mari, N. M., de Puig, H., Huang, A., Lee, R., Slomovic, S., Galbersanini, T., Lansberry, G., Sallum, H. M., Zhao, E. M., Niemi, J. B., & Collins, J. J. (2021). Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nature Biotechnology, 39, 1366–1374.https://doi.org/10.1038/s41587-021-00950-3
Where a cell free system with LacZ and CPRG was successfully designed as a wearable colourmetric bio-sensor.
However, I am a bit stumped on how to create a plasmid design for a cell free system and if the promoters I am using for the NahR operon are compatible with a cell free system as they are from Pseudomonas putida not Ecoli (would it work with Ecoli RNA polymerase or is there such thing as using Pseudomonas putida RNA polymerase in CFPS?). Is this a possible next step or is my goal not compatible?? Is there a way to optimise what I am doing so it would work??
Thank you if you read this far!! :)