Semi-synthetic minimal cells as a tool for biochemical ICT

Pasquale Stano, Giordano Rampioni, Paolo Carrara, Luisa Damiano, Livia Leoni, Pier Luigi Luisi

University of Roma & University of Bergamo, Italy

BioSystems, 109 (2012) 24 –34

Presented by:Avinash Chandra KushwahaM.Sc. |st Year

Aim – To use Semi–Synthetic Minimal Cells (SSMCs) as artificial entities able to communicate, by processing Biochemical Information, with natural systems.

Introduction Biological systems evolved with the ability to communicate with their biotic surroundings through

chemical signalling. Production, perception and decoding of the information carried by signal molecules allow

individuals of a community to interact, cooperate, and coordinate their activities, establishing complex social behaviours.

The production of functional proteins in SSMCs raises the possibility to generate semi synthetic cell-like systems expressing the biochemical apparatus for signal molecules production, perception and decoding.

The construction of synthetic systems capable of communicating with natural living organisms would greatly impact the applications of synthetic biology and biochemical-based information and communication technologies (ICTs) in medical sciences, for example for smart programmable and drug-producing systems.

SSMCs can find application is the development of a new information and communication technology (ICT) paradigm, based on molecular communication.

Keywords Synthetic biology Minimal Cells Liposomes Autopoiesis ICT Quorum sensing

The Theory Of Autopoiesis

The distinctive property of living systems is autopoiesis (namely, “self-production”), that is, the capability of these systems of producing and maintaining themselves – their material identity.Autopoiesis is a global property of living systems, which relies not on their physico-chemical components taken separately, but in the way in which these components are organized within the systems.Autopoiesis offers three theoretical tools to synthetic biology: • A definition of life, providing the description of a mechanism able to generate

minimal living systems.• A theoretical characterization of these systems’ cognitive interaction with the

environment.• A theory of communication between minimal living systems.

Semi-Synthetic Minimal Cells (SSMCs)

SSMCs are cell-like structures assembled in the laboratory starting from a minimal set of molecular compounds such as enzymes, nucleic acids, ribosomes inside lipid vesicles (liposomes).

By studying the SSMC model, a deep understanding on various fundamental questions in origin of life research can be obtained, such as solute encapsulation, internal reaction, self-reproduction, selection/competition.

The minimal set of 208 genes (which is environment-dependent) represents the number of housekeeping genes essential to define an autonomous living system from the viewpoint of genomics.

SSMCs can synthesize nucleic acids and express proteins & enzymes. SSMCs can synthesize receptors, molecular motors (like FoF1-ATP synthase) and

import/export protein channels.

SSMCs: Two broad design classes

First, enzymes are encapsulated inside liposome at the aim of performing specific task. • DNA template, deoxyribonuleotides and DNA polymerases (multiple copies of DNA

sequences obtained).• Four enzymes can synthesize lipid, phosphatidylcholine (for growth of membrane).• For RNA synthesis, by DNA template, ribonucleotides & RNA polymerase.

Second, the cell-free transcription/translation machinery is encapsulated inside liposomes together with a DNA gene, so that it produces a functionally active protein.

• A PURE system is composed of four modules: transcription, translation, tRNAs charging with amino acids, and energy recycling.

• Incorporation of the PURE system into liposomes, fulfils the aspects of synthetic biology and minimal life.

Molecular communication in biological systems

The communication in biological world is mainly based on molecular signals. The information-carrier molecule (or signalling molecule) must physically move from the sender to the

receiver. Five logically distinguishable processes can be identified:

(1) encoding the message in form of a molecule, (2) sending the molecule,

(3) propagation/transportation, (4) receiving the molecule, (5) decoding the message

source

transmission

channel destination

/function

encoding

transmission reception

decoding

sender receiver

Noise interference(environment)

Bacterial communication and quorum sensing

Quorum Sensing (QS), a cell-cell signalling system that allows a bacterial population to co-ordinately reprogram gene expression in response to cell density.

The QS-response is achieved when the concentration of the signal molecule produced and secreted by bacteria reaches a threshold level, corresponding to a certain cell density, the “quorum”.

QS is also involved in the regulation of a wide variety of physiological processes including antibiotic biosynthesis, motility, plasmid conjugal transfer, biofilm formation, and the production of bacterial virulence factors in plant, animal and human pathogens.

Fig - Structure of representative bacterial QS signal molecules.

Acylated Homoserine Lactones (AHLs) are QS molecules in Gram-negative bacteria.Small peptides are QS molecules in Gram-positive bacteria.Autoinducer-2(Al-2) is a third class of QS molecule in both types.

Some of them are also used to exchange information between bacteria of different species or genera occupying the same ecological niche, and even to interact with their eukaryotic hosts

Requirements for QS modules

1. Synthesized in a simple enzymatic reaction;2. Freely diffuse outside the producer;3. Be stable in the medium while propagating between the producer and the

receiver;4. Be easily detected by the receiver;5. Carry a message that is easily transduced and detected.

The QS systems based on Acylated Homoserine Lactones (AHLs) provide promising examples of biological communication modules transferable to SSMCs technology.

Synthetic cell/Natural cell communication

Three relevant issues related to the communication ability of a synthetic cell-like system:1. Synthetic cell, in order to be defined truly alive, should also acquire the

capacity of chemical communication.2. Advancement in cell-free synthetic biology and bottom-up bioengineering.3. Future potentially practical applications based on our capacity of controlling

the communication between synthetic and natural cells.

By advancing the current biotechnology for SSMCs construction, medical potential applications of more sophisticated, communicating and programmable synthetic cells can be envisaged. A goal would be to construct cell-like systems that, once injected in human body, reach a specific target region and thanks to chemical information processing, is able to act accordingly, for example producing in situ a cytotoxic drug or a stimulus to trigger a cellular response.

target tissue

addressing/targeting

transport

)in/out(

self-destructing

scaffold

sensorial

system

encapsulated

enzymatic

system

Fig: Pseudofactories or nanofactories.

Experiments & Results

Semi-synthetic minimal cells

The “droplet transfer” method for producing SSMCs

PURE system kit (Protein synthesis Using Recombinant Elements)

Productions from PURE system(cell-free system)

Water soluble proteins/enzymes, Green Fluorescent Protein (GFP) T7 RNA polymerase β-galactosidase Qβ-replicase β- glucoronidase Water insoluble enzymes: two Acyltransferases GPAT & LPAAT. A pore-forming α-hemolysin also produced inside a liposome by cell-free system.

α-Hemolysin molecules self-assemble as a heptamer on the lipid membrane, creating a pore which allowed the free passage of small molecules (<3 kDa) from the environment to the liposome and vice versa.

Key Experiment

Liposome-based synthetic cells encapsulate the chemical components that give rise to the “formose” reaction, produce linear and branched carbohydrates from formaldehyde.

Furanose products could escape from the synthetic cells via α-hemolysin pore, react with borate ions, resulting furanosyl boronates which is structurally very similar the QS signal molecule AI-2.

Al-2 like compounds activated the expression of the QS, generating a synthetic entity which is able to send a signal to a natural receiver.

The use of SSMCs with incorporating a variety of genetic/regulatory and metabolic modules responsible for signal synthesis and signal perception, synthetic cell can establish a two-way communication system with natural cells.

chemical

communication

chemical

communication

semi-synthetic minimal cells

DNA

ribosome s

enzymes

liposome

t-RNAs

Results

Living Cells

Communication among Synthetic Systems & Living Systems

Concluding remarksBio-chemical ICTs are a new class of technologies, often bio-inspired, aimed to expand our knowledge and capacities of manipulating the structural and functional information present in the molecular and supramolecular worlds.Minimal cell-like ones are particularly interesting systems for interfacing with bio-systems in a programmable way.In more general terms, implicitly points to the use of synthetic cells as computational tools.Based on transcription/translation reactions inside liposomes – a new bio-chem ICT can be developed in near future, based on the chemical communication among synthetic cells and natural cells

Thank You!