week-09-hw-cell-free-systems

Week 9: Cell-Free systems!

Part A: General and Lecturer-Specific Questions

General questions:

1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production.

Cell-free protein synthesis (CFPS) offers important advantages over traditional in vivo expression because it provides a more open, flexible, and controllable reaction environment. Since there is no living cell to maintain, the researcher can directly adjust variables such as ionic strength, pH, redox conditions, DNA template concentration, cofactors, chaperones, detergents, lipids, or energy substrates without worrying about cell viability. CFPS is also typically faster, allowing protein production in hours rather than requiring cell growth, transformation, and induction steps over longer periods. In addition, it facilitates rapid prototyping of constructs and reaction conditions (Garenne et al., 2021; Jewett et al., 2008).

Another major advantage is that CFPS is particularly useful for proteins that are difficult to express in living cells, such as toxic proteins, membrane proteins, or proteins that require non-standard reaction environments. Because the system is open, reagents can be supplied directly and problematic cellular responses such as toxicity, growth inhibition, or proteolytic stress can be reduced (Garenne et al., 2021; Meyer et al., 2025).

Two cases where cell-free expression is more beneficial than cell-based production are:

2. Describe the main components of a cell-free expression system and explain the role of each component

A cell-free expression system generally includes the following components:

References 1. (Garenne et al., 2021); 2. (Jewett et al., 2008); 3. (Caschera, 2025); 4. (Harris et al., 2020); 5. (Meyer et al., 2025).

Additionally, a view of a CFPS by the article:

Figure 1. CFPS compounds from (Hong et al., 2014)

3. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

Energy regeneration is critical in CFPS because transcription and translation consume large amounts of ATP and GTP. Without a continuous energy supply, the reaction quickly slows or stops, lowering protein yield. In addition, some simple high-energy substrates can accumulate inorganic phosphate, which chelates magnesium and impairs ribosomal activity, further reducing productivity (Yavad et al., 2025). One way to ensure continuous ATP supply is to use an ATP-regeneration system based on phosphoenolpyruvate (PEP), which donates phosphate groups for ATP resynthesis. Another effective strategy is to use maltodextrin/polyphosphate-based metabolism in crude extracts, which can support longer-lasting and more cost-effective ATP regeneration through endogenous metabolic enzymes (Caschera & Noireaux, 2015; Chen et al., 2019).

Method	Paper
An alternative approach is the use of metabolic energy regeneration systems, such as glucose-based pathways. Anderson et al. (2015) demonstrated that glucose metabolism in eukaryotic cell-free systems enables sustained ATP production through endogenous enzymatic pathways, improving reaction longevity and cost efficiency.	Figure 2. Abstract from: Anderson et al., 2015)

Additionally, recent tools, such as ATP biosensors (Mu et al., 2024), provide insights into the energetic dynamics of biological systems. Although not directly used for ATP regeneration, these biosensors can help optimize cell-free reactions by monitoring ATP availability in real time and guiding adjustments in energy supply strategies.	Figure 3. Abstract from: (Mu et al., 2024)

4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

In the following table, a comparison is made between different cell systems and the cell-free expression.

Table 1: Comparison of prokaryotic versus eukaryotic cell-free expression systems

Description: The table information was based on (Garenne et al., 2021; Jewett et al., 2008; Meyer et al., 2025), and (Fenz et al., 2014) for the eukaryotic field.

5. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.

A rational way to design a cell-free experiment to express a membrane protein would be to optimize both the biochemical reaction conditions and the DNA construct. Based on the VDAC study by Zayni et al. (2021), I would use a prokaryotic cell-free expression system and focus particularly on the region surrounding the translation start site, since the paper showed that expression efficiency was mainly governed by translation initiation and mRNA conformation near the start codon, rather than by differences in transcription. (Zayni et al., 2021)

Steps:

1. I would first select a plasmid, in this case a plasmid + T7 promoter and a properly positioned Shine-Dalgarno sequence.
2. Then, I would evaluate the 5’ UTR and the first codons of the translated region. (Based on the paper of Zayni, it demonstrated that these sequence elements strongly influence whether the ribosome can properly dock and initiate translation) (Zayni et al., 2021)
   1. It is important to analyze the accessibility of the ribosome docking site and estimate the ΔEopen of the mRNA around the start codon.
   2. A lower ΔEopen would indicate that the ribosome-binding region is more accessible and therefore more likely to support efficient protein expression.

Optimization

To optimize the construct, I would test the following different design strategies:

• Adjusting the spacing between the Shine-Dalgarno sequence and the start codon.
• Adding a translation enhancer if native expression is too weak.
• If I want to preserve the native amino acid sequence, introducing synonymous mutations (Substitutions; DNA changes that alter a codon’s nucleotide sequence but not the resulting amino acid) (Oelschlaeger, 2024) in the first several codons to reduce the inhibitory mRNA secondary structures without altering the protein itself.

This last strategy in the paper aims to substantially improve VDAC expression while preserving the WT protein sequence.

Main challenges

The main challenges in this setup could be:

1. Low translation efficiency: Caused by poor ribosome access to the start region.
2. Non-native N-terminal additions: Enhancers like His-tags or CAT- derived sequences are used.
3. Persistent low expression of membrane proteins, since these proteins are inherently difficult to produce.

I would address these by first identifying whether the limitation is transcriptional or translational. Since the paper showed that mRNA levels remained similar across poorly and well-expressed constructs, I would prioritize troubleshooting the translation-initiation region rather than assuming transcription is the problem. I would then redesign the coding sequence near the start codon to improve RDS accessibility, ideally using synonymous codon optimization.

6. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.

If I observed a low yield of my target membrane protein in a cell-free system, three possible reasons would be:

Case 1. Poor translation initiation due to inhibitory mRNA secondary structure:

Case 2. Suboptimal construct architecture near the 5′UTR and RBS

Case 3. Inadequate reaction conditions for cell-free synthesis

Additionally:

Figure 4. Abstract from Zayni et al., 2021	This paper does not mainly optimize membrane insertion conditions such as lipid composition or detergents; rather, it shows that for this membrane protein, low expression was strongly linked to mRNA structure and translation initiation, and that in silico sequence optimization can significantly improve yield.

Click here to download the paper: Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence-An Experimental Study of the Human Voltage-Dependent Anion Channel

Homework question from Kate Adamala

Based on my final individual project, KitBi, I am looking to detect early Gram-negative bacteria biofilms from kitchen surfaces and utensils in an easy, portable, economic, and quick method similar to a pH paper.

1. Pick a function and describe it.

a. What would your synthetic cell do? What is the input, and what is the output?

• My synthetic cell would detect quorum-sensing molecules from Gram-negative bacteria before mature biofilm formation and convert that signal into a visible reporter output.
• Input: AHL molecules released by Gram-negative bacteria.
• Output: fluorescence or colorimetric signal produced by the synthetic cell. AHLs are a practical early target because they are extracellular signals associated with quorum sensing in Gram-negative bacteria, and quorum sensing is closely tied to virulence and biofilm-related behaviors.

(Lentini et al., 2014; Miller & Gilmore, 2020; Galloway et al., 2010)

b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

Yes, theoretically. There are published cell-free biosensors for quorum-sensing molecules, so detection itself does not strictly require encapsulation. A paper by Wen et al. describes a cell-free biosensor for quorum-sensing biomarkers in infectious disease contexts.

(Wen et al., 2017)

Additionally, talking about the membrane, it gives more of an artificial cell logic, similar to Kate’s example: the vesicle becomes a defined sensing unit, can protect the Tx/Tl mix, and about selective exchange with the environment. Reviews on synthetic cell–living cell communication also support liposome-based systems as useful platforms for chemical communication.

(Mukwaya et al., 2021; Rampioni et al., 2019)

c. Could this function be realized by a genetically modified natural cell?

Yes. Natural bacterial biosensors for AHLs exist, and whole-cell AHL sensor systems are well established.

(Kumari et al., 2006)

d. Describe the desired outcome of your synthetic cell operation.

In the presence of Gram-negative quorum-sensing signals, the synthetic cell turns on a reporter and gives an early warning that a biofilm-prone bacterial population may be emerging on the surface being tested. This follows the objectives of detecting early and intervening before eradication becomes harder. The review on quorum-sensing molecule detection explicitly frames QS signals as potentially useful early diagnostic indicators.

(Miller & Gilmore, 2020)

2. Design all components that would need to be part of your synthetic cell.

a. What would the membrane be made of?

The membrane will be a phospholipid membrane with cholesterol, for example, POPC + cholesterol. Since this is a standard and defensible artificial-cell style membrane in liposome-based systems. Also, based on Lentini’s example, it used phospholipids plus cholesterol as a simple artificial-cell membrane concept.

(Lentini et al., 2014)

b. What would you encapsulate inside? Enzymes, small molecules.

Inside the vesicle, I would encapsulate:

• a bacterial cell-free Tx/Tl system
• a DNA circuit containing an AHL-responsive transcription factor
• a reporter gene such as sfGFP or lacZ

This is realistic because cell-free AHL biosensing has already been demonstrated, and bacterial lysate-based cell-free systems are commonly used for such biosensors

(Wen et al., 2017; Didovyk et al., 2017)

c. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian)

A bacterial system, ideally E. coli-based, is the most reasonable choice here, because AHL quorum-sensing modules like LuxR/plux are bacterial and do not require a mammalian expression background.

(Wen et al., 2017)

d. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

It will follow the communication logic:

Gram-negative bacteria on a surface release AHLs → AHL diffuses into or across the synthetic cell membrane → AHL binds its regulator inside the vesicle → reporter gene turns on.

(Ding et al., 2014; Mukwaya et al., 2021)

3. Experimental details

a. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)

For my project, I decided to focus on a liposome-based synthetic cell encapsulating an E. coli cell-free expression system and a LuxR-responsive reporter circuit to detect AHL molecules released by Gram-negative bacteria as an early warning of biofilm development.

Lipids:

• POPC
• Cholesterol

Genes:

• luxR
• sfGFP under a LuxR-activated promoter such as PluxI

(Lentini et al., 2014; Miller & Gilmore, 2020)

b. How will you measure the function of your system?

Fluorescence output from GFP, measured by plate reader, microscopy, or bulk fluorescence.

(Wen et al., 2017)

Homework question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field — Architecture, Textiles/Fashion, or Robotics — and propose an application using cell-free systems that are functionally integrated into the material. Answer each of these key questions for your proposal pitch:

For this section, I am exploring the fashion/textile option with UV clothes based on: (Lawrynowicz et al., 2024; Sąsiadek-Andrzejczak & Kozicki, 2023)

• Write a one-sentence summary pitch sentence describing your concept.

A sun-responsive shirt embedded with freeze-dried cell-free systems that activate under UV exposure, and produce a visible color change to reduce heat absorption and signal high solar intensity.

• How will the idea work, in more detail? Write 3-4 sentences or more.

The textile would contain microencapsulated, freeze-dried cell-free systems embedded within its fibers that respond to UV radiation. Upon exposure to sunlight, the system becomes activated (for example: heat or humidity) and produces a colorimetric output, where higher UV intensity generates a deep purple pigment, while lower exposure results in a softer pastel tone. This gradient response allows the textile to visually indicate different levels of solar exposure rather than a simple on/off signal. As a result, the material functions both as a real-time UV indicator and as an adaptive aesthetic element in fashion.

• What societal challenge or market need will this address?

This system addresses increasing UV exposure and heat stress, especially in regions like Latin America, especially here in Ecuador, where solar radiation is intense due to altitude.

High UV exposure is linked to skin damage and long-term health risks, but people often lack real-time awareness of exposure levels. A responsive textile could act as a personal UV sensor, helping individuals make better decisions about sun protection while also improving comfort and awareness.

• How do you envision addressing the limitations of cell-free reactions (e.g., activation with water, stability, one-time use)?

Table 2. Addressing limitations of cell-free systems

Homework question from Ally Huang

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space!

For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/ .

1. Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words)

Biofilms are a real challenge in spacecraft because microbes can colonize surfaces and water systems, threatening hardware reliability and potentially crew health. NASA documents note that biofilm formation has been observed in ISS systems, including water lines, where it contributed to clogging and pump issues. This is significant for humanity because long-duration missions will require reliable, low-resource methods for monitoring contamination. It is also scientifically interesting because microgravity and spaceflight conditions can alter microbial behavior, including biofilm-related traits.

(Ravichandran et al., 2025)

Recommended lectures from NASA

Figure 5. Microbial Research Guide from Colorado et al., 2021	Figure 6. Conference of 2022 about Redefining Microbiological Risk Mitigation During Spaceflight from Ott, 2022

Link to access the following documents in the Sources section

2. Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words)

Biofilm- and quorum-sensing-related RNA targets from Gram-negative bacteria, such as luxS, lasI/lasR, or pslA transcripts

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

Biofilm formation does not begin as a visible layer; it starts with changes in gene expression and cell-cell signaling. Quorum-sensing and biofilm-associated transcripts can therefore serve as early molecular indicators of biofilm development before major fouling occurs. Detecting these RNA targets would help identify when bacteria are shifting from planktonic growth toward surface-associated communities, which is exactly the stage where intervention is most useful in spacecraft systems with limited maintenance capacity.

(Flores et al., 2024; Vélez et al., 2023)

4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

I hypothesize that Gram-negative bacteria exposed to space-relevant stress conditions will show an increased abundance of quorum-sensing or biofilm-associated RNA targets compared with non-biofilm controls, and that these changes can be detected using a compact toolkit that combines miniPCR, BioBits®, and fluorescence readout.

• My goal is to develop an early-warning molecular screening strategy for spacecraft biofilm risk. The reasoning is that freeze-dried cell-free systems are portable and low-resource, while the Genes in Space toolkit already includes fluorescence-based tools designed for constrained environments.

If successful, this approach could support routine monitoring of microbial contamination during long-duration missions.

5. Outline your experimental plan - identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words)

I would test RNA extracted from Gram-negative bacterial cultures grown under a biofilm-promoting condition versus planktonic control cultures. miniPCR would amplify cDNA corresponding to selected biofilm-related targets, and BioBits® plus the P51 fluorescence viewer would be used to visualize signal output. Controls would include a no-template negative control and a non-biofilm bacterial condition. Data would consist of fluorescence presence or relative intensity across samples, indicating whether biofilm-associated targets are enriched under spacecraft-relevant stress conditions.

(Jung et al., 2023)

Part B: Homework Part B: Individual Final Project

Title:

KitBi: An Early-Warning Fluorescent Biosensor for Early Biofilm Commitment on Food-Contact Surfaces

Aims!

Aim 1 — Experimental aim

• Design and computationally validate a PcsgD-driven fluorescent reporter construct in non-pathogenic E. coli K-12 to detect early biofilm-inducing physiological states relevant to food-contact surfaces.

Aim 2 — Development aim

• Test the reporter under controlled early-attachment conditions using simulated or future experimental comparisons across planktonic, surface-exposed, and stainless-steel-associated growth states, and optimize the system with an internal normalization module or alternative promoters such as PcsgBAC.

Aim 3 — Visionary aim

• Translate KitBi into a portable early-warning platform for food-contact surface monitoring, potentially through freeze-dried, paper-based, or cell-free-compatible readouts that support preventive hygiene decisions before mature biofilm establishment.

Final Slide

Coming soon!

Weekly reflection:

This week made me realize how universal biofilms are. I initially thought of them mainly in food and clinical contexts, but learning about their presence in space environments really changed my perspective. It was surprising to see that biofilms can form even under microgravity conditions and still represent a risk for systems like water lines and surfaces in spacecraft.

This reinforced the relevance of my project, since early detection is not only important on Earth but also in highly controlled environments like space missions. It made me think that KitBi could have broader applications beyond food safety, especially in settings where prevention is critical and intervention is limited.

Thank you for reading! I updated this entry also on my personal Notion webpage. To check it, please enter here! Notion W9

References and sources

PART A:

General Questions:

Caschera, F. (2025). Cell-free protein synthesis platforms for accelerating drug discovery. Biotechnology Notes, 6, 126-132. https://doi.org/10.1016/j.biotno.2025.02.001

Caschera, F., & Noireaux, V. (2015). A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. Metabolic Engineering, 27, 29-37. https://doi.org/10.1016/j.ymben.2014.10.007

Chen, J., Mitra, R., Zhang, S., Zuo, Z., Lin, L., Zhao, D., Xiang, H., & Han, J. (2019). Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism. Applied and environmental microbiology, 85(19), e00984-19. https://doi.org/10.1128/AEM.00984-19

Chipman, D. M., Woolley, A. C., Chau, D. N., Lance, W. A., Talley, J. P., Green, T. P., Robbins, B. C., & Bundy, B. C. (2025). Cell-Free Protein Synthesis Reactor Formats: A Brief History and Analysis. SynBio, 3(3), 10. https://doi.org/10.3390/synbio303001

Fenz, S. F., Sachse, R., Schmidt, T., & Kubick, S. (2013). Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes. Biochimica Et Biophysica Acta (BBA) - Biomembranes, 1838(5), 1382-1388. https://doi.org/10.1016/j.bbamem.2013.12.009

Garenne, D., Haines, M.C., Romantseva, E.F. et al. Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). https://doi.org/10.1038/s43586-021-00046-x

Harris, N.J., Pellowe, G.A. & Booth, P.J. Cell-free expression tools to study co-translational folding of alpha helical membrane transporters. Sci Rep 10, 9125 (2020). https://doi.org/10.1038/s41598-020-66097-4

Jewett, M.C., Calhoun, K.A., Voloshin, A. et al. An integrated cell‐free metabolic platform for protein production and synthetic biology. Mol Syst Biol 4, MSB200857 (2008). https://doi.org/10.1038/msb.2008.57

Meyer, C., Arizzi, A., Henson, T. et al. Designer artificial environments for membrane protein synthesis. Nat Commun 16, 4363 (2025). https://doi.org/10.1038/s41467-025-59471-1

Oelschlaeger P. (2024). Molecular Mechanisms and the Significance of Synonymous Mutations. Biomolecules, 14(1), 132. https://doi.org/10.3390/biom14010132

Yadav, S., Perkins, A. J. P., Liyanagedera, S. B. W., Bougas, A., & Laohakunakorn, N. (2025). ATP Regeneration from Pyruvate in the PURE System. ACS synthetic biology, 14(1), 247–256. https://doi.org/10.1021/acssynbio.4c00697

Zayni, S., Damiati, S., Moreno-Flores, S., Amman, F., Hofacker, I., Jin, D., & Ehmoser, E. K. (2021). Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence-An Experimental Study of the Human Voltage-Dependent Anion Channel. Membranes, 11(10), 741. https://doi.org/10.3390/membranes11100741

Kate Adamala:

Didovyk, A., Tonooka, T., Tsimring, L., & Hasty, J. (2017). Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synthetic Biology, 6(12), 2198-2208. https://doi.org/10.1021/acssynbio.7b00253

Ding, Y., Wu, F., & Tan, C. (2014). Synthetic Biology: A Bridge between Artificial and Natural Cells. Life, 4(4), 1092-1116. https://doi.org/10.3390/life4041092

Galloway, W. R. J. D., Hodgkinson, J. T., Bowden, S. D., Welch, M., & Spring, D. R. (2010). Quorum Sensing in Gram-Negative Bacteria: Small-Molecule Modulation of AHL and AI-2 Quorum Sensing Pathways. Chemical Reviews, 111(1), 28-67. https://doi.org/10.1021/cr100109t

Kumari, A., Pasini, P., Deo, S. K., Flomenhoft, D., Shashidhar, H., & Daunert, S. (2006). Biosensing Systems for the Detection of Bacterial Quorum Signaling Molecules. Analytical Chemistry, 78(22), 7603-7609. https://doi.org/10.1021/ac061421n

Lentini, R., Santero, S., Chizzolini, F. et al. Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour. Nat Commun 5, 4012 (2014). https://doi.org/10.1038/ncomms5012

Miller, C., & Gilmore, J. (2020). Detection of Quorum-Sensing Molecules for Pathogenic Molecules Using Cell-Based and Cell-Free Biosensors. Antibiotics, 9(5), 259. https://doi.org/10.3390/antibiotics9050259

Mukwaya, V., Mann, S. & Dou, H. Chemical communication at the synthetic cell/living cell interface. Commun Chem 4, 161 (2021). https://doi.org/10.1038/s42004-021-00597-w

Rampioni, G., D’Angelo, F., Leoni, L., & Stano, P. (2019). Gene-Expressing Liposomes as Synthetic Cells for Molecular Communication Studies. Frontiers in bioengineering and biotechnology, 7, 1. https://doi.org/10.3389/fbioe.2019.00001

Wen, K. Y., Cameron, L., Chappell, J., Jensen, K., Bell, D. J., Kelwick, R., Kopniczky, M., Davies, J. C., Filloux, A., & Freemont, P. S. (2017). A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples. ACS Synthetic Biology, 6(12), 2293-2301. https://doi.org/10.1021/acssynbio.7b00219

Peter Nguyen

Lawrynowicz, A., Vuori, S., Palo, E., Winther, M., Lastusaari, M., & Miettunen, K. (2024). Transforming fabrics into UV-sensing wearables: A photochromic hackmanite coating for repeatable detection. Chemical Engineering Journal, 494, 153069. https://doi.org/10.1016/j.cej.2024.153069

Sąsiadek-Andrzejczak, E., & Kozicki, M. (2023). Multi-Color Printed Textiles for Ultraviolet Radiation Measurements, Creative Designing, and Stimuli-Sensitive Garments. Materials, 16(16), 5622. https://doi.org/10.3390/ma16165622

Ally Huang

Flores, P., Luo, J., Mueller, D. W., Muecklich, F., & Zea, L. (2024). Space biofilms - An overview of the morphology of Pseudomonas aeruginosa biofilms grown on silicone and cellulose membranes on board the international space station. Biofilm, 7, 100182. https://doi.org/10.1016/j.bioflm.2024.100182

Jung, J. K., Rasor, B. J., Rybnicky, G. A., Silverman, A. D., Standeven, J., Kuhn, R., Granito, T., Ekas, H. M., Wang, B. M., Karim, A. S., Lucks, J. B., & Jewett, M. C. (2023). At-Home, Cell-Free Synthetic Biology Education Modules for Transcriptional Regulation and Environmental Water Quality Monitoring. ACS synthetic biology, 12(10), 2909–2921. https://doi.org/10.1021/acssynbio.3c00223

Ravichandran, V., Krishnan, B., Tinwala, M., Kumar, A. S., & Jobby, R. (2025). Microbial resilience in space: Biofilms, risks and strategies for space exploration. Life Sciences In Space Research, 47, 1-13. https://doi.org/10.1016/j.lssr.2025.05.004

Vélez Justiniano, Y. A., Goeres, D. M., Sandvik, E. L., Kjellerup, B. V., Sysoeva, T. A., Harris, J. S., Warnat, S., McGlennen, M., Foreman, C. M., Yang, J., Li, W., Cassilly, C. D., Lott, K., & HerrNeckar, L. E. (2023). Mitigation and use of biofilms in space for the benefit of human space exploration. Biofilm, 5, 100102. https://doi.org/10.1016/j.bioflm.2022.100102

SOURCES:

Image PART A:

Hong, S. H., Kwon, Y. C., & Jewett, M. C. (2014). Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis. Frontiers in chemistry, 2, 34. https://doi.org/10.3389/fchem.2014.00034

Figure 5 link: https://www.nasa.gov/wp-content/uploads/2021/10/microbial_research_2021_tagged.pdf

Figure 6 link: https://ntrs.nasa.gov/api/citations/20220008788/downloads/Redefining Microbiological Risk Mitigation during Spaceflight.pdf