Bacterial Dirigibles

DIVISION OF BACTERIOLOGY AND MYCOLOGY, IVRI
Presented By:
Sangram P. Ramane
PhD Scholar
Roll no. 1447

OUTLINE
Introduction
Background
Concept of Bacterial Dirigible
Bacterial Dirigible Properties
Targeted Delivery
Designing Genetic Circuits
Biological Nanofactories
Applications
Conclusion
Slide no. 2BACTERIAL DIRIGIBLES

INTRODUCTION
BACTERIAL DIRIGIBLES
 Microorganisms programmed using synthetic constructs of DNA commands
(Voigt 2006).
 Emerging applications of synthetic biology: To design bacteria to produce
therapeutic agents, Use of live bacteria as targeted delivery systems (Garmory
et al 2003, Martin et al 2003).
 Genome contains commands dictating how cells
eat, reproduce, communicate, move and interact
with their environment.
 Genome manipulations with greater achievements
were started in 1970s.
 Genetic engineering is entering a new era of
synthetic biology i.e. engineering of biology.
Slide no. 3

BACKGROUND
BACTERIAL DIRIGIBLES Introduction continued… … Slide no. 4

BACTERIAL DIRIGIBLES Introduction continued… …
Synthetic biology combines science and engineering with common goal of design and
construction of new biological functions.
Slide no. 5

BACTERIAL DIRIGIBLES Introduction continued… …
Synthetic biology combines science and engineering with common goal of design and
construction of new biological functions.
Computerengineering
Biologicalengineering
A possible hierarchy for synthetic biology is inspired by computer engineering
(Andrianantoandro et al 2006)
Slide no. 6

CONCEPT OF BACTERIAL DIRIGIBLES
 Unique concept at 241st National Meeting &
Exposition of the American Chemical Society
(ACS 2011)
 "We're building a platform that could allow
bacteria to be the next-generation disease
fighters“ - William E. Bentley.
 New candidate for treating diseases ranging
from food poisoning to cancer termed “Bacterial
Dirigibles“
 Modified bacteria have look of blimps (non-rigid
floating dirigible/airship).
BACTERIAL DIRIGIBLES Slide no. 7

 Reprograms bacteria
 Produce antibiotics,
insulin, and other
medicines and materials
 Bacteria grow in nutrient
solutions in enormous
stainless steel vats in
factories.
 Release antibiotics or
insulin into vats
 Harvest the medicine
 Processing
 Eventual use in people
Traditional genetic engineering
BACTERIAL DIRIGIBLES Concept of bacterial dirigibles continued… … Slide no. 8

Swallowed or injected
into the body
Bacteria travel
to the diseased
tissue
Start producing substances
To fight the disease
Give the bacteria a
biochemical
delivery address,
which is the locale
of the disease
Bacterial dirigible approach takes bioengineering a step further
BACTERIAL DIRIGIBLES Slide no. 9Concept of bacterial dirigibles continued… …

BACTERIAL DIRIGIBLES PROPERTIES
Designed
“Genetic circuit”
Genetic circuit
that acts as
“Nanofactory”
Autonomously
navigates
Systematically
transport
Strategically
distribute
medicine “cargo”
Bacteria
programmed
Produce the
disease-fighting
chemicals at
destination
Rather than creating a drug in a lab to be used as medicine, this new
research is a way of repurposing bacteria as attack against diseases.
Property of targeting, sensing and switching capabilities
Bacterial dirigibles properties continued… … Slide no. 10

Designed
“Genetic circuit”
Genetic circuit
that acts as
“Nanofactory”
Autonomously
navigates
Systematically
transport
Strategically
distribute
medicine “cargo”
Bacteria
programmed
Produce the
disease-fighting
chemicals at
destination
Rather than creating a drug in a lab to be used as medicine, this new
research is a way of repurposing bacteria as attack against diseases.
Property of targeting, sensing and switching capabilities
Bacterial dirigibles properties continued… … Slide no. 10

TARGETED DELIVERY BY BACTRIA
 Targeted delivery investigated for treatment of HIV (Pollock et al
2008), cancer (Torchilin 2008), diabetes (Morishita et al 2008),
pulmonary diseases (Durcan et al 2008), endothelial diseases
(Simone et al 2008), etc.
 Selective targeting can be effected by quantum dots (Weng et al
2008), viral vectors (Wang et al 2008), nanoparticles (Murphy et al
2008), liposome (Meers et al 2008), polymers (Xiong et al 2009), etc.
 Increase effect of the delivered drug to targeted area and reduces
unwanted and non-specific effects.
 Ultimate goal is to alter the response in the targeted area to a
‘desired state’ which is different from the ‘original state’.
BACTERIAL DIRIGIBLESBACTERIAL DIRIGIBLES Slide no. 11

1
2
3
BACTERIAL DIRIGIBLES Targeted delivery by bacteria continued… … Slide no. 12

BACTERIAL DIRIGIBLES Targeted delivery by bacteria continued… …
DESIGNING GENETIC
CICUITS
Slide no. 13
Ligand-Receptor combination with engineered biological switch can effectively produce
drug only at the needed location.
Ligand-
Receptor
combination
Required
drug
synthesis

DESIGNING GENETIC CIRCUITS
Inhibition
site
Enhancer
site
Promoter
site
Gene information
DNA DNA
 Bacteria control of gene expression through
complex cellular networks (Barkai et al 1997).
 These networks are known to consist of various
regulatory modules (Becskei et al 2000).
 With these basic regulatory modules and motifs,
artificial networks that mimic natural expression
are constructed (Sprinzak et al 2005).
TYPICAL GENETIC CIRCUIT
Slide no. 14

Typical process for engineering gene circuits
BACTERIAL DIRIGIBLES Designing genetic circuits continued… …
DNA manipulations done in a ‘copy, cut
and paste’ manner using polymerases,
restriction endonucleases and ligases
Animal experiment and observes their
dynamics in living cells
A genetic circuit is constructed from a strand of DNA: genes, promoters, operator sites, RBS and terminators.
By using different mathematical tools,
algorithms and computer software
A working design usually requires
multiple rounds
1
2
3
4
(Marguet et al 2007)
Slide no. 15

BIOLOGICAL NANOFACTORIES
A nanometer sized biological factory
 Chitosan based or Antibody based
or Whole Bacterium can also be
act as nanofactory.
 Comprises multiple functional
modules attached together.
 One Module = One genetic circuit
 Each module performing different
function.
 When deployed, attach to the
targeted cells, produce molecules
of interest.
 Autoinducer-2 based gene
expression to alter cellular
response.
(LeDuc et al 2007)
BACTERIAL DIRIGIBLES Slide no. 16

AI-2 Biological nanofactory based bacterial communication and cellular
response alteration for expression of gene of interest
 Quorum sensing: Bacteria exchange small chemical molecules,
monitor their population density and co-ordinate gene expression in
a population dependent manner (Fuqua et al 1994).
 Occurs within a species (intra-species), with other species (inter-
species) and with other organisms (inter-kingdom) (Waters et al
2005).
 Communication molecules: Acylated homoserine lactones (AHL),
oligopeptides, autoinducer-2 (AI-2), pseudomonas quinolone signal
(PQS) and bradyoxetin (Lowery et al 2008).
 Relative ubiquity of AI-2, it has been called as the “USM i.e. Universal
Signalling Molecule”.
Biological nanofactories continued… … Slide no. 17

AI-2
biosynthesis
pathway is
reconstructed
in vitro in a
biological
nanofactory
BACTERIAL DIRIGIBLES Biological nanofactories continued… … Slide no. 18
Autoinducer-2 Based
Biological Nanofactories (Fernandes et al 2008)

Lsr Transporter
AI-2
AI-2
 Autoinducer-2 (AI-2)
 4,5-dihydroxy-2,3-pentanedione (DPD)
 S-adenosyl methionine (SAM)
 S-adenosyl homocysteine (SAH)
 S-ribosylhomocysteinase (LuxS)
 S-adenosylhomocysteine nucleosidase (Pfs)
AI-2 concentration
exceeds given threshold
Biological nanofactories continued… …
Gene of interested
downstream of Lsr genes
Slide no. 19
AI-2
AI-2 Biological nanofactory based bacterial
communication and cellular response
alteration for expression of gene of interest

1. Synthesis of the magnetic carrier by co-precipitation of iron salts and chitosan
2. Attachment of Pfs and LuxS to carrier by ‘activation’ using tyrosinase
3. Capture of target cells by the magnetic nanofactories
4. Recovery of captured cells using an external magnet
5. Cell surface synthesis and delivery of AI-2 by enzymes Pfs and LuxS
6. Uptake of AI-2 and production of cellular response
Overview of assembly of
Magnetic Biological Nanofactories
BACTERIAL DIRIGIBLES Biological nanofactories continued… … Slide no. 20
(Fernandes et al 2008)

BACTERIAL DIRIGIBLES Biological nanofactories continued… …
Overview of assembly of
Antibody Biological Nanofactories
Slide no. 21
(Fernandes et al 2008)

DESTROY HERBICIDE
REPAIR COCRETE
Bacteria with property of targeting, sensing and switching capabilities
Bacterial dirigible
NEXT GENERATION APPLICATIONS
ENVIRONMENTAL
Slide no. 22

Bacteria with property of targeting, sensing and switching capabilities
Bacterial dirigible
NEXT GENERATION APPLICATIONS
DISEASE FIGHTERS
AGAINST IBD
AGAINST CANCER CHOLERA TREATMENT
Next generation applications continued… … Slide no. 23

GMB programmed to
swim down fine cracks
in the concrete.
Very
specific
pH of
material
CaCO3 and a
bacterial glue
which would
combine with
the filamentous
bacterial cells
Bacillus
subtilis1
2
3
4
BACTERIA TO REPAIR CONCRETE
BACTERIAL DIRIGIBLES Next generation applications continued… … Slide no. 24

 Atrazine, one of the most
common contaminants found
in water supplies.
 Bacteria are programmed by
using synthetic aptamer
riboswitches.
 Engineered E. coli with atzA
from Pseudomonas sp. which
encodes a chlorohydrolase to
that converts atrazine to
hydroxyatrazine.
Little RNA
pieces that can
bind directly to
a ligand or
signal molecule
and cause a
change in gene
expression and
motility
REPROGRAMMING BACTERIA TO SEEK AND
DESTROY HERBICIDE
BACTERIAL DIRIGIBLES Next generation applications continued… …
(Sinha et al 2010)
Slide no. 25

Finding RNA
sequences that
would bind to
atrazine
Successful binders
and tested for
riboswitch activity
Engineer E. coli
1
2
3
Riboswitch atzA
Chlorohydrolase4

 Human growth factors are potential
therapeutic agents for various
inflammatory disorders of GIT.
 But, unstable when administered
orally and systemic administration
requires high doses increasing the
risk of unwanted side effects.
 GM Bacteroides ovatus deliver KGF-2
when exposed xylan, directly to the
damaged cells of gut.
GENETICALLY ENGINEERED BACTERIA ARE SWEET SUCCESS
AGAINST INLFAMATORY BOWEL DISEASE
(Hamady et al 2010)
Slide no. 27

PROGRAMMED BACTERIA IS ON ITS WAY FOR
TREATING CHOLERA
 Programmed E. coli produce signaling molecules recognized by V. cholerae.
 Prevent the V. cholerae from producing toxins that cause infection.
(Ruder et al 2011)
Slide no. 28

 Researchers used E. coli as vehicles, to
deliver a cytotoxic chemical that kills
tumour sites.
 Engineered E. coli comprised of
multiple genes, that contain the
instructions for the bacteria to carry
out assignment.
 Each genetic device specifies the
what, when, where, and how of each
step that the E. coli must execute to
ultimately kill tumour sites.
E. Coli: The Cure for Cancer

Bacteria would be
injected into a patient’s
bloodstream where it
would to carry out
three main tasks:
Avoid the immune
system,
Find and invade the
tumour site
Release the cytotoxin
to kill the cancer cells
Slide no. 30

Avoid the immune system
Bacteria would be
injected into a patient’s
bloodstream where it
would to carry out
three main tasks:
Avoid the immune
system,
Find and invade the
tumour site
Release the cytotoxin
to kill the cancer cells
Slide no. 30

Injected into the
bloodstream of a
cancer patient
Travel to the tumour
site sense hypoxic
regions
Bind to surface proteins
called β1-integrins on the
cancer cells
1
2
3
4
5
Once inside the tumour cell, the bacteria
would deliver a cytotoxin to the
cytoplasm of the tumour cell
Kill the cancer cell
Slide no. 31
Invading of
tumour site and
killing tumour
cells

Invading of tumour site and killing tumour cells
Viable tumour (green/blue),
vasculature (red) and bacterial
(orange/yellow) signals are
visualised.
Whole Body luminescence
demonstrating co-localisation
of bacteria (orange) and
subcutaneous tumour (green).
(Cronin et al 2012)
Slide no. 32

CONCLUSION
Synthetic biology is attributed to rewire cell's genetic circuits for synthesis of
novel products.
Artificial genetic circuits that made bacteria with targeting, sensing & switching
capabilities.
Nanofactory could produce chemical signals that trigger cells to synthesize
natural disease-fighting substances.
A innovative view makes use of the reprogrammed bacterial cell as the
therapeutic product.
Approach could be used to treat cancer, infection, diabetes and a wide range of
diseases caused by the lack of a certain protein.
‘Bacterial dirigibles' emerge as next-generation disease fighters.
Slide no. 33

BACTERIAL DIRIGIBLES, By - Sangram P Ramane, VBM, IVRI (1st Yr PhD). Monday, 14.05.2012 The End

Bacterial Dirigibles

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