Week 9 HW: Cell-Free Systems

Part A: General and Lecturer-Specific Questions

general questions

Advantages of cell-free protein synthesis (CFPS) over in vivo methods

Mainly, there is no living cell and no cell membrane involved, and so any component of the reaction can be added or removed in the course of the controlled reaction. There are fewer variables to control compared to an experiment and synthesis in a living cell. No transformation process is involved, and so the reaction is faster. Cell-free is more beneficial when a toxic protein is synthesized or a protein that incorporates non-canonical amino acids.

Main components of a cell-free expression system

Main components: cell extract, ribosomes, tRNAs, aminoacyl-tRNA synthetases, factors for elongation, initiation, and release, a template, NTPs, amino acids, energy regeneration system, and a buffer.

Lysate: a lysate is an unfractionated content of the cytoplasm, it supplies ribosomes, tRNAs, aminoacyl-tRNA synthetases, elongation and initiation factors, and the metabolic enzymes.

Buffer: a buffer needed to keep pH for the optimal work of transcription and translation enzymes by counteracting accumulating acidic byproducts of energy metabolism. Potassium Glutamate provides potassium as main cation ions cells are adapted to, and glutamate is E.coli’s major cytoplasmic anion modulating osmolarity, and so both are recreating the native ionic environment. Potassium is needed for ribosomes to fold and assemble (by neutralizing negative phosphate charges on rRNA and allowing ribosomal subunits to not repel) and for translation itself, but too high concentration inhibits translation (promotes dissociation of the ribosomal subunits and initiation complex destabilize) as many of the interactions involved in the process are reversible electrostatic; glutamate unlike chloride is large and it does not interact with protein surfaces with positively charged regions and functional interfaces. Magnesium Glutamate provides magnesium ions as the major cofactors for polymerases, it defines the structure and function of ribosomes (neutralizes negative phosphate charges of rRNA), it stabilises nucleotides and RNA (binds to phosphates of nucleotides, mRNA, tRNA); and glutamate is a safe (large) counter-ion maintaining osmolarity as in potassium glutamate. Potassium phosphate monobasic/dibasic provides an additional buffer system, and so may add the capacity to counteract pH drift from accumulating acetate. Phosphate serves as an energy metabolism substrate and regenerates ATP and nucleotide triphosphates, but too much phosphate can diminish magnesium (as the magnesium salt is insoluble) and inhibit the reaction.

Energy and nucleotides: Ribose replenishes the NTP pool in the system, where nucleoside monophosphates and free guanine are supplied instead of ready-made triphosphates. Glucose is the primary energy source and substrate for glycolysis to generate ATP for transcription and translation. Acetate produced as a byproduct shifts the pH to acidic. AMP, CMP, GMP, UMP are ribonucleoside monophosphates phosphorylated by kinases in the lysate, precursors to ATP, GTP, CTP, UTP for mRNA synthesis. Guanine converts to guanine nucleotides through the purine salvage pathway, it replenishes GTP pool that is consumed in transcription and translation.

Amino Acids: 17 Amino Acid Mix, Tyrosine, and Cysteine are building blocks for proteins. Tyrosine is added separately because of its different solubility (in high pH), and cysteine oxidizes quickly.

Additives: Nicotinamide is a precursor for NAD⁺/NADH indispensable for glycolysis; these are not consumed in transcription or translation. Nuclease Free Water dissolves all the components, is deprived of RNases or DNases that would degrade the template and mRNA.

The importance of energy regeneration and a method for continuous ATP supply

Energy equivalents are indispensable for chemical bonds to form and transcription and translation to occur. A secondary energy sources (phosphoenolpyruvate, creatine phosphate) along with the corresponding kinases can be added to regenerate ATP.

Prokaryotic vs. eukaryotic cell-free systems

Prokaryotic systems give high yields, they are cheaper and more scalable; eukaryotic are slower in their kinetics, more expensive and give lower yields. An antibody fragment or a protein that requires posttranslational modifications of multi-domain folding would require the eukaryotic systems; a small protein not requiring modifications would fit into a prokaryotic system.

An experiment for a membrane protein

Micelles or liposomes can be added into the mix to provide a hydrophobic support to hydrophobic domains of transmembrane proteins so that they don’t aggregate and misfold. An experiment would require selecting (screening) the best hydrophobic supplement, optimizing temperature and cation concentrations to achieve the optimal time for proper folding, optimizing concentrations and membrane insertion factors of chaperons if necessary. Folding needs to be verified with downstream assays involving binding analysis, activity assays, and aggregation assays.

Addressing the low protein yield problem

One reason could be a bad template design, a degraded template, or a non-optimized template; the template can be verified on a gel. Another reason could be not enough energy equivalents present in the mix; energy substrates can be added or the reaction can be supplied with a system that replenishes the substrates and removes byproducts that inhibit the reaction (continuous-exchange). Another reason can be too fast synthesis kinetics that does not allow proper folding or an environment that favors aggregation or degradation; reaction temperature can be lowered, chaperons and protease inhibitors can be added.

question from Kate Adamala

Function: sensing cortisol in wearable cortisol patch for hours-long continuous monitoring. A microfluidic sweat collector built with lipid-vesicle-encapsulated TX-TL with a glucocorticoid receptor would collect sweat and output a reporter fluorescent protein as the result of cortisol binding to its receptor.

Could cell-free Tx/Tl alone do this? Yes, with riboswitches described below, but a patch with a minimal cell system would be more selective and pharmacologically relevant.

Could a genetically modified natural cell do this? Yes

Desired outcome: sweat passively crosses the vesicle bilayer and cortisol binds to the receptor, and reporter synthesis starts.

Components include a membrane and the encapsulated contents. The latter includes a eukaryotic cell-free Tx-Tl system (HeLa lysate) with chaperones, human glucocorticoid receptor, a template for a chromoprotein under a specific promoter, energy equivalents, and kinases.

Source of the Tx/Tl system: HeLa cells.

Communication with the environment: cortisol will passively cross the membrane as it’s hydrophilic.

Experimental details Lipids: POPC, Cholesterol, DOPG, DOPE, Atto647N-DOPE, Trehalose Genes: GRE-driven reporter (mNeonGreen)

Measuring the function: reading mNeonGreen fluorescence with single-vesicle imaging, in dose-response experiments addressing the sensor’s dynamic range covering physiological levels of cortisol. Affinity and selectivity measurements are required.

questions from Peter Nguyen

Disposable nitrile gloves with freeze-dried cell-free biosensors that react to cortisol levels by changing colour and supply the information about animal’s physiological state at the time of handling.

How would it work: a glove material contains micro wells with lyophilized cell-free transcription-translation machinery and genetic circuits. A riboswitch responsive to cortisol would trigger the expression of a chromoprotein. Rodent urine or sweat released during animal handling would activate the reaction reservoirs and a reporter in it, causing the colour to change on the glove surface within minutes.

Societal challenge or market need: animal handling that is part of drug testing or neuroscience studies is frequently unstandardized, although it defines animal physiology and behaviour, and efficient techniques to handle animals gently, without triggering a stress response or for quick mitigation of a stress response are available. These gloves would feel a demand for basic science, conservation biologists, and animal welfare auditors for a non-invasive and real-time stress and health assay.

The limitations: 1) a liquid activating the reaction may not be sufficient or optimal; 2) reaction reagents may lose stability over time; 3) the sensitivity for sweat cortisol and urine cortisol needs to be very different, so a large dynamic range has to be factored into the design.

question from Ally Huang

Hippocampal neural stem cells are exceptionally radiation-sensitive; their loss during spaceflight underlies loss of new neurons that is tied to behavioural flexibility, persistent cognitive decline, memory, and mood deficits documented in rodents exposed to X-rays, gamma-rays, and heavy particles. As Mars class missions are planned by multiple companies and governments, and the missions exceed current chronic exposure limits, understanding how cosmic radiation damages neurogenic gene templates is critical for the health of astronauts. Radiation damage to DNA templates encoding neurogenic genes directly reduces protein output, providing an adequate model of the molecular vulnerability driving astronaut cognitive decline, and cell-free TX-TL systems serve as faithful molecular biosensors.

The target: A linear or plasmid template with the human BDNF coding sequence fused to sfGFP. The target and the challenge: BDNF controls neuronal maturation dependent on synaptic plasticity and neural stem cell proliferation, and its levels are decreased in irradiated hippocampus; however, the integrity of its template has not been studied sufficiently yet. Radiation-induced lesions on the BDNF template in a cell-free model may impair transcription and translation.

Hypothesis: Space radiation will produce dose-dependent damage to the BDNF-sfGFP template that significantly reduces cell-free protein output relative to ground controls, with the longer BDNF–sfGFP construct showing greater vulnerability per reaction than the shorter sfGFP-only control.

Reasoning: Damage probability increases with template length, radiation exposure time, and the mode of pre-exposures, and so longer transcripts are more likely to carry lesions or mutations that block transcription and translation. Demonstrating time and dose-dependent vulnerability of the BDNF template under real space flight conditions would provide evidence that BDNF is affected in space.

Experimental plan: Samples would include the BDNF-sfGFP template, an sfGFP-only template with a length and GC-content matching the first template. Both will be aliquoted as dried DNA. Samples pre-irradiated with low and moderate doses of gamma-rays and samples left on Earth must be included. MiniPCR® thermal cycler can be used to detect template degradation. sfGFP can be quantified with imaging (P51® viewer).

Part B: Individual Final Project

The final project slide was put in the slide deck. A mock DNA order was placed.