Barcoding biological reactions with DNA-functionalized vesicles

Peruzzi, Justin A.; Jacobs, Miranda L.; Vu, Timothy; Kamat, Neha P.

doi:10.1101/672287

Cited by 7 publications

(9 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Genes listed in SI Table S2 were ordered as gene blocks and cloned into a high copy plasmid used in previous work ( 39 ). Different fusion proteins were generated using standard restriction enzyme cloning techniques using Phusion DNA polymerase and restriction enzymes from Thermo Fisher.…”

Section: Methodsmentioning

confidence: 99%

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

Peruzzi

Steinkühler

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The extent to which membrane biophysical properties, such as hydrophobic thickness, can drive membrane protein organization remains unknown. Inspired by this question, we used de novo protein design, molecular dynamic simulations, and cell-free systems to elucidate how membrane-protein hydrophobic mismatch affects protein integration and organization in synthetic lipid membranes. We found that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We used this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to self-organizing membrane-based materials with new functions.

show abstract

Section: Methodsmentioning

confidence: 99%

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

Peruzzi

Steinkühler

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…15 The fusion was also demonstrated using DNA tags, with proposed mechanism similar to that of SNARE proteins: the fusion tags facilitate outer leaflet mixing, followed by inner leaflet mixing, and finally mixing of the lumen of the liposomes. 16 For our goal of building combinatorial libraries of synthetic minimal cells and constructing genetic pathways with multiple nodes, a highly controllable and programmable fusion system was required. This directed us toward the use of nucleic acid membrane tags, rather than small molecules or lipid additives that act nondiscriminatively on all membranes present in the sample.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Programmable Fusion and Differentiation of Synthetic Minimal Cells

et al. 2022

View full text Add to dashboard Cite

Synthetic cells can mimic the intricate complexities of live cells, while mitigating the level of noise that is present natural systems; however, many crucial processes still need to be demonstrated in synthetic cells to use them to comprehensively study and engineer biology. Here we demonstrate key functionalities of synthetic cells previously available only to natural life: differentiation and mating. This work presents a toolset for engineering combinatorial genetic circuits in synthetic cells. We demonstrate how progenitor populations can differentiate into new lineages in response to small molecule stimuli or as a result of fusion, and we provide practical demonstration of utility for metabolic engineering. This work provides a tool for bioengineering and for natural pathway studies, as well as paving the way toward the construction of live artificial cells.

show abstract

“…Later, DNA emerged, which due to its higher stability assumedly became the predominant genetic material 37, 39 . In synthetic biology, RNA and DNA have been utilized to perform cell-free gene expression 40 inside lipid vesicles 11, 41 aiming at pathways to the bottom up construction of a living cell.…”

Section: Resultsmentioning

confidence: 99%

Liposome-assisted in-situ cargo delivery to artificial cells and cellular subcompartments

Xue

Stephenson

Gözen

2022

Preprint

View full text Add to dashboard Cite

We report on liposome-mediated targeted delivery of membrane-impermeable constituents into surface-adhered giant lipid compartments, employed as artificial cells. Soluble cargo compounds are delivered by means of an open-space microfluidic device, which perfuses selected lipid compartments with loaded small unilamellar vesicles (SUVs) composed of cationic lipids. The SUV membranes fuse with the surface-adhered containers, merging their contents. We monitored the fusion process via Förster resonance energy transfer (FRET) by labeling both the membranes of the SUVs and the target compartments with a fluorophore pair. We established that, upon fusion, water-soluble dyes, fluorescently labeled genetic polymers, sugars and proteins carried by the SUVs can be successfully internalized at high yield. Finally, by transferring carbonic anhydrase (CA) to the giant lipid compartments, enzymatic hydrolysis of the prefluorescent carboxyfluorescein diacetate (CFDA) is demonstrated by the emission intensity increase emanating from the product carboxyfluorescein (CF). Spontaneous subcompartmentalization occurred during liposomal delivery of the enzyme, leading to CF formation in an organelle-like subcompartment. The reported targeted delivery technique enables chemical reactions and cell-free gene expression in synthetic cell models with unprecedented ease and precision, and opens pathways to protocell architectures with distinct functional subcompartments in the context of origins of life research.

show abstract

Barcoding biological reactions with DNA-functionalized vesicles

Cited by 7 publications

References 70 publications

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

Programmable Fusion and Differentiation of Synthetic Minimal Cells

Liposome-assisted in-situ cargo delivery to artificial cells and cellular subcompartments

Contact Info

Product

Resources

About