1) Semi-synthetic minimal cells (SSMCs) encapsulating biochemical components can produce and perceive signal molecules, allowing them to communicate with natural cells. 2) An experiment showed that SSMCs performing the formose reaction could produce carbohydrate molecules that activated quorum sensing in natural bacteria, demonstrating communication. 3) SSMCs hold potential as programmable synthetic systems that could one day interface and exchange information with living organisms through biochemical signaling, representing a new paradigm for biochemical information and communication technology.

1. 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 Kushwaha
M.Sc. |st Year

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

3. 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.

4. Keywords
 Synthetic biology
 Minimal Cells
 Liposomes
 Autopoiesis
 ICT
 Quorum sensing

5. 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.

6. 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.

7. 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.

8. 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)

9. 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.

10. 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

11. 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.

12. 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.

13. 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.

14. Experiments & Results

15. Semi-synthetic minimal cells

16. The “droplet transfer” method for producing SSMCs

17. PURE system kit (Protein synthesis Using Recombinant Elements)

18. 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.

19. 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.

20. chemical
communication
chemical
communication
semi-synthetic minimal cells
DNA
ribosome s
enzymes
liposome
t-RNAs
Results
Living Cells
Communication among Synthetic Systems & Living Systems

21. Concluding remarks
Bio-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

22. Thank You!