Week 9: Cell Free Systems

General questions

(Question 1) Since the cell-free protein synthesis system eliminates the cell membrane, this means that the environment that the reaction is performed in is less limited by what can enter or exit the cell as it alters the dependence of the reaction on other cellular constraints. For example, the energy source and the chaperone/cofactor concentrations can be altered independently of the cell’s own needs. This poses a particularly interesting environment for cases such as the incorporation of non-standard amino acids, in which cells may not contain the machinery necessary to incorporate but contain machinery that would resist the incorporation of such amino acids. Another intriguing application would be the prototyping of vaccine antigen production. Due to the speed that cell-free systems can perform at, the system would be able to produce a functional antigen from a gene sequence much quicker without the need to engineer a stable cell line to express the desired antigen.

(Question 2)

• The cell extract provides the basic machinery of a cell, such as the ribosomes, translation factors, endogenous tRNA synthetases, chaperones and folding machinery, as well as the RNAP.
• The DNA/mRNA template determines what will be expressed by the cell-free system
• the RNAP allows the template to be transcribed
• Amino acids allow dor the translation of the template
• The energy regeneration system provides energy for the translation process and sustains the reaction
• The NTPs are used for translation elongation and mRNA synthesis
• Mg2+ ions are necessary for ribosome assembly and K+ ions stabilize the ribosome and supports translation fidelity
• Cofactors can also be added depending on the target protein

(Question 3) Energy provision regeneration is critical in cell-free systems due to the very reason it is cell-free, in the sense that there is no longer a cell to produce the energy for the reactions. Since transcription and translation are energy-intensive processes, a cell-free system requires the supplementation of energy in order to successfully complete it’s assigned task. One method to continuously supply ATP to a cell-free experiment is using a phosphocreatine/creatine kinase system. Creatine kinase drives the phosphorylation of ATP by transferring the phosphate group from phosphocreatine to ADP, thus producing creatine.

(Question 4) Prokaryotic and eukaryotic cell-free expression systems differ by the machinery that is present in the native cells. In prokaryotic cells, transcription and translation are coupled and occur nearly simultaneously whereas in eukaryotic systems, transcription and translation are separated by location within the cell. Eukaryotic systems are also able to perform post-translational modifications, whereas prokaryotic systems are not equipped for these kinds of modifications.

I chose to explore the production of a T4 lysozyme within a prokaryotic cell-free system, as it is typically toxic to the bacterial host through the degradation of the peptidoglycan cell wall, however, the cell-free system bypasses this constraint.

Due to eukaryotic cell-free systems’ unique ability to perform post-translational modifications, I wanted to explore the hormone erythropoietin. The glycosylation is an essential part of the hormone’s production, which would be bypassed within a prokaryotic cell-free system.

(Question 5) For the synthesis of membrane proteins using the cell-free system, one of the main limitations would be that the hydrophobic nature of the membrane protein would be produced within an aqueous mix, directly opposing the very nature of the molecule. For this reason, the experimental setup would require a hydrophobic carrier/chaperone in order to ensure that the hydrophobic protein can be synthesized properly. To achieve this, I researched that nanodiscs would be the best way to achieve this, and are in fact used for functional studies of membrane proteins [1].

(Question 6)

1. One potential reason for low yield of a target protein could be that there is not enough supplemented energy in order to carry out the entire transcription/translation process, as discussed in question 3. In order to troubleshoot this, it would be beneficial to increase the phosphocreatine concentration, implement a feeding strategy, or switch energy systems.
2. Another potential reason for low ield could be that there is inefficient translation of the target protein, which can occur when the mRNA is degraded faster than it can be translated. Some potential troubleshooting strategies could include codon optimizing the sequence, to optimize the template concentration, or to add an RNase inhibitor to prevent the mRNA degradation.
3. An additional reason that there may be low yield of a target protein could be contributed to protein misfolding. In order to troubleshoot this, you could supplement chaperones into the cell-free system.

Kate Adamala’s questions

For this assignment, I wanted to toy with the concept of using a cell-free system for an idea similar to my final project rather than engineering the fibroblasts themselves for an application to surgical response . For this, I want to utilize the cell-free system to sense and respond to an inflammatory environment appropriately.

(Question 1)

In this concept, the synthetic cell would be designed as an inflammation-responsive growth factor delivery system for chronic wound healing. Chronic wounds are characterized by their failure to heal due to unresolving inflammation, caused by persistently elevated MMPs that degrade growth factors quicker than the tissue can respond to them, resulting in dysfuntion in the balance between pro- and anti-inflammatory signals. For my synthetic system, I want it to sense the elevated MMP-9 activity within the wound microenvironment, releasing the encapsulated template DNA of the growth factor, PDGF-BB, allowing it to reach therapeutically relevant concentrations.

Without encapsulation, the cell-free machinery would be degraded by the immune mediators within the wound environment and the template DNA would be degraded by extracellular DNases. The encapsulation of the template DNA also allows for the sense-and-respond mechanism to be functional.

While this could be replicated in genetically modified cells, there are a few pros to using a synthetic system instead. For example, the synthetic cell is less likely to cause an immune response and due to the lack of a cell itself, cannot replicate and therefore is less likely to become tumorigenic.

Ideally, the synthetic cell would remain undetectable whenever MMP-9 is low, only triggered in a wound environment where the MMP-9 concentration is elevated, indicating a wound that is unable to heal. When this happens, the MMP-9 would cleave a crosslinker which releases the DNA template and initiates the trancsiption and translation process. This allows the production and release of PDGF-BB into the wound environment, eventually resulting in the recruitment and activation of local fibroblasts, contributing to the wound healing process. As the healing progresses, the MMP-9 levels would normalize, initiating the negative feedback of the PDGF-BB production.

(Question 2)

For the membrane of the synthetic cell, I need something that is biocompatible, can respond to the MMP trigger, and has enough stability to survive in a wound environment until the input/output system is triggered. For this, I anticipate using DOPC, DOPE, DOPG, and cholesterol as the main components of the membrane and including a MMP-responsive crosslinker, such as GPLGIAGQ, which is a well-validated MMP-9 cleavage substrate [2].

As for within the membrane, I would plan to encapsulate the cell-free transcription/translation machinery as well as the template DNA for PDGF-BB under the control of a T7 promoter. To aid with the proper folding of PDGF-BB, I would also need the caperones DsbC and a glutathione buffer due to the presense of critical disulfide bonds. I think it would also be helpful to include an RNase inhibitor to protect the mRNA from any RNase activity.

Since the PDGF-BB is not dependent on post-translational modifications, it would be alright to use an E. coli cell-free sytem due to the lower costs and higher yield output.

The signal input of the system would rely on MMP-9 senseing, which would not need a membrane channel since it depends on the cleavage of the peptide crosslinker. As for the output, the PDGF-BB release would primarily rely on the destabilization of the membrane through the cleavage of the cross-linker, which is a form of passive release. However, this could be optimized using a pore-forming mechanism, such as an alpha-hemolysin channel. The small molecules involved within the cell-free mixture (NTPs, amino acids, and Mg2+), would not be able to freely pass through the DOPC bilayer, however since all the transcripition/translation machinery is encapsulated, external substrate uptake would not be required and naturally limits the operational window.

(Question 3)

Lipids

• DOPC
• DOPE
• DOPG
• Cholesterol
• GPLGIAGQ conjugate

Genes

• PDGFB (human, but codon-optimized for E. coli) for therapeutic output
• dsbC for correct disulfide bond formation in PDGF-BB
• hlyA for membrane pore (PDGF-BB release)
• T7 RNAP

In order to measure the function of the system, we would first need to validate the function of the transcription/translation machinery through the confirmation of PDGF-BB expression using SDS-PAGE and western blot. The SDS-PAGE could also be used in order to validate whether or not the PDGF-BB disulfide bonds were folded correctly. It would also be necessary to validate the vesicle formation through the use of dynamic light scattering. Finally, the validation of the MMP-9 triggered release would also be neceassary to ensuring the proper functioning of the systme. By using the vesicles to encapsulate florescence, by adding MMP-9, which is supposed to cleave the GPLGIAGQ cross-linker, the presence of fluorescence would validate this encapsulation.

Peter Nguyen’s question -> Fashion/Textile

Body odor has long been the subject of self-consciousness, and with this project, a wearable fabric embedded with cell-free systems senses the skin’s biochemistry in real time and responds accordingly with various harmonious fragrances, masking the need to feel embarrassed.

The skin’s chemical landscape is constantly changing throughout our daily lives. Sweat can cause the pH to swift between 4.5 and 7.5, depending on exercise, stress, and metabolism. Skin temperature can fluctuate several degrees depending on the the area and throughout the day. These physiological signals often go undetected, but this project will actively interpret those signals as inputs to a fragrance synthesis program embedded within the fabric itself.

When I imagine this system, I imagine three different circuits existing along the fabric.

• The first would be a floral base that exists at the resting body physiology, which is typically pH 5.5-6.5. Using a pH-sensitive promoter, the enzyme linalool synthase would be produced, and GPP would be encapsulated as well. Upon the conversion of GPP, it would emit a floral scent that would act as an ambient perfume [3].
• The second layer would be activated from a rise in body temperature, often achieved during physical activity. This would reequire a temperature-sensitive switch that would activate the enzyme limonene synthase, which would also convert GPP into limonene, a citrus scent in order to mask the increase in body odor [4].
• The final layer would be sweat activated, which can result after exercise or from sterss, causing a rise above normal pH. Upon the activation of a high pH-sensitive switch, the expression of valencene synthase would be activating, producing valencene, which provides a woody smell [5].

One of the limitations of using cell-free systems is premature water activation from rain, humidity, or other sources. In order to prevent this, we could employ a double layer membrane which consists of a hydrophobic outer cell and a pH-responsive polymer for the inner shell, requiring a certain pH to activate the cell-free system. Another potential limitation could be the limited use due to the energy and precursor consumption of the circuit. In order to bypass this, the capsules should be loaded with material that is able to perform the reaction many times for a set number of wears.

Ally Huang’s questions

During long periods of microgravity, muscles become atrophied due to the lack of resistance. Even on short 5-11 day missions, astronauts have lost up to 20% of muscle mass, with current countermeasures such as resistance exercise and nutrition guidelines, rely on verbal communication of problems rather than obervation of physiological biomarkers [6][7]. By the time atrophy becomes clinically apparent, irreversible damage could’ve already occurred [8]. On a Mars mission lasting 2-3 years, undetected progressive atrophy could result in crewmembers becoming physically unable to perform critical operations [9]. Real-time monitoring is therefore not a convenience, but a critical safety measure that should be invested in.

For this project, I would target myostatin (GDF-8) protein concentration and IL-6 myokine levels in saliva, which could be detected with a toehold switch coupled to a fluorescent reporter output.

Myostatin is a protein that firectly suppresses muscle growth, and elevated myostatin signals active muscle catabolism. IL-6 is released by contracting and stressed muscle fibers, which can indicate productive exercise response but elevated IL-6 can also indicate inflammatory muscle breakdown. These two targets can provide a picture of whether muscle tissue is in a productive remodeling state or in a catabolic degenerative state. Both proteins can be detected within the saliva, allowing for non-invasive sampling.

I hypothesize that the salivary myostatin and IL-6 profiling using BioBits freeze-dried cell-free toehold switch biosensors would be able to detect the molecular signature of muscle degradation at least two weeks prior to clinically measurable muscle volume loss. I reason that because molecular changes precede gross anatomical changes in all known muscle wasting conditions, early molecular detection is both scientifically justified and clinically actionable.

The weekly saliva collection will be processed as follows.

1. Add 5uL salivea to two BioBits freeze-dried toehold switch reaction tubes (one for myostatin and one for IL-6)
2. Rehydrate with 45uL of nuclease-free water
3. Incubate for 37 C for 2 hours
4. Read the fluorescent output using P51 viewer

It would be necessary to obtain a pre-flight baseline from each crewmember. For positive control, a synthetic myostatic/IL-6 spike at a known concentration could be used and for negative control, using water only.

Individual final project