Endosynbiology Endosynbiology The use of engineered bacterial - - PowerPoint PPT Presentation

Endosynbiology Endosynbiology The use of engineered bacterial endosymbionts to create synthetic

• rganelles in host cells.

ENDOSYMBIOSIS

ancient heterotrophic proteobacteria mitochondria protection nutrients

ENDOSYMBIOSIS

Pea aphid Nutrient-poor diet of sap Bacteriome housing endosymbionts

APPLICATIONS

autoimmune

Many disorders can be attributed to a dysfunctional or absent gene or gene product

cancers

siRNA silencing of oncogenes for cancer therapy Monoclonal antibody therapy targeting autoreactive antibodies

OUTLINE

making endosymbionts the pHlow system

regulation containment

biosafety symbiont candidates and future work

MAKING ENDOSYMBIONTS

how to turn a free-living bacterium into an endosymbiont:

Facilitate entry into cell Ensure stable survival and replication Design secretion of useful product

A PRE-EXISTING SYSTEM

Warsaw 2009 & 2010 designed a protein delivery system utilising Invasin and Listeriolysin O.

Surface protein derived from Yersinia enterocolitica. A secreted hemolysin derived from Listeria monocytogenes.

Invasin (INV) Listeriolysin O (LLO

HOW THE SYSTEM WORKS

• 1. Invasin binds to β1-

integrins

• 2. Endosymbiont is

endocytosed

• 3. Listeriolysin breaks

down the endosome

• 4. Entry is complete

(1) (2) (3) (4)

pHLOW- MEDIATED ENTRY

pH INDUCIBLE PROMOTERS

1.

• 1. E. coli
• E. coli acid shock

acid shock response esponse (BBa_K1170000) 2.

• 2. L.
• L. lactis

lactis p170 p170 (BBa_K1677300) 3.

• 3. L.
• L. lactis

lactis p170-CP25 p170-CP25 hybrid hybrid (BBa_K1677301)

as the pH in the endosome drops, it induces expression of listeriolysin O

CRE-LOX RECOMBINATION

RNA PSEUDOKNOTS

RNA secondary structures between ORFs cause translational delay BBa_K1677391: IBV pseudoknot

Design:

• Start codons were removed from viral sequences
• 20bp were added upstream and downstream of the

pseudoknots

• Placed outside of ORFs

BBa_K1677369: MMTV pseudoknot BBa_K1677743: TGV pseudoknot BBa_K1677666: HCV229E pseudoknot

STRUCTURAL MODELLING

Structures were predicted using DotKnot and visualised using PseudoViewer.

MMTV 1x Global MMTV 3x Global

Sperschneider, J., Datta, A., DotKnot: pseudoknot prediction using the probability dot plot under a refined energy model. Nucleic acids research 2010, 38, e103. Sperschneider, J., Datta, A., Wise, M. J., Heuristic RNA pseudoknot prediction including intramolecular kissing hairpins. Rna 2011, 17, 27-38.

STRUCTURAL MODELLING

TGV 3x Global TGV 1x Global

Structures were predicted using DotKnot and visualised using PseudoViewer.

TOXIN-ANTITOXIN SYSTEM

Isolated from P . aeruginosa Antitoxin binds toxin preventing cell death Prevents: Escape of recombinant bacteria Horizontal Gene transfer

With no IPTG With IPTG

CONTAINMENT

• 1. arabinose-dependent expression of invasin
• 2. constitutive expression of toxin
• 3. IPTG-dependent expression of antitoxin
• 4. Cre-lox deletion of invasive genes

BIOSAFETY

ENDOSYMBIONTS

• Lactic acid bacteria model organism
• Gram positive + anaerobic
• Low immunogenicity + anti-oxidative ability
• Part of healthy human microbiome
• Generally Regarded As Safe (GRAS)

BBa_K1677103: LysM-INV BBa_K1677101: LLO (codon (codon optimised

• ptimised)

)

ENDOSYMBIONT CANDIDATE 1: LACTOCOCCUS LACTIS

ENDOSYMBIONT CANDIDATE 2: SYNECHOCYSTIS PCC6803

• Freshwater cyanobacteria

model organism

• 1-2 day generation time
• Previous evidence of survival

Integrative plasmids psbA2 and pCPC

Agapakis, C. M., Niederholtmeyer, H., Noche, R. R., Lieberman, T. D., Megason, S. G., Way, J. C., & Silver, P. A. (2011). Towards a synthetic chloroplast. PLoS One, 6(4), e18877.

Synechocystis PCC6803 Lactococcus lactis Escherichia coli K12

Sources: http://veja0.abrilm.com.br/assets/images/2011/5/38595/Escherichia-coli-surto-alemanha-20110530-size-598.bmp , http://w3.unisa.edu.au/ciam/groups/bg/images/synechocystis.jpg,, http://textbookofbacteriology.net/Lactococcusnewtdb.jpg , http://www.atcc.org/~/media/Attachments/F/1/2/8/1768.ashx,

HelaT CHO-7 BHK21

FUTURE WORK

population control transmission during cell division cell specific targeting post- translational modifications

quorum sensing mitochondrial model

• ther invasin/

adhesin proteins coexpression

• f enzymes

HUMAN PRACTICES

• Biosafety
• Endosymbiont choice
• Communication strategy
• Pseudoknots and intellectual property
• Hosted Aussie iGEM meet up
• Outreach projects
• Make your own GMO high school workshop

(collaboration with UNSW Aspire and UNSW Biomod team)

• BABS UNSW ethics debate

• Used previously existing biobricks (INV and LLO) to design a

more efficient and safe cellular entry system (pHlow)

• Improved characterisation of two previously existing biobricks

(CP44 and ASR promoter)

• Submitted and characterised new pH inducible promoter (L.

lactis P170)

• Submitted three individual pseudoknot biobricks and

conducted structural modeling

• Submitted three pseudoknots downstream from IPTG-

inducible promoters

• Invented new containment mechanism (toxin + anti-toxin)
• Submitted a complete cellular entry system for Lactococcus

(LysM-INV + LLO)

• Adapted integrative plasmids to be biobrick compatible
• Optimised Lactococcus and Synechocystis transformation

protocols

ACHIEVEMENTS

QUESTIONS?

Anaer Anaerobic

• bic

Aer Aerobic

• bic

Heter Heterotr

• trophic
• phic

Autotr Autotrophic

• phic
• E. coli

x x

• L. lactis

x x Synechocystis x x x

ENDOSYMBIONT 1:

• E. COLI
• Ubiquitous synthetic biology

chassis

• Comparability to previous

research (Agapakis, 2011)

• Poor public perception
• 20 minute generation time

Agapakis, C. M., Niederholtmeyer, H., Noche, R. R., Lieberman, T. D., Megason, S. G., Way, J. C., & Silver, P. A. (2011). Towards a synthetic chloroplast. PLoS One, 6(4), e18877.

ENTEROINVASIVE E. COLI STRAINS

Synechocystis PCC6803 Lactococcus lactis Escherichia coli K12

Sources: http://veja0.abrilm.com.br/assets/images/2011/5/38595/Escherichia-coli-surto-alemanha-20110530-size-598.bmp , http://w3.unisa.edu.au/ciam/groups/bg/images/synechocystis.jpg,, http://textbookofbacteriology.net/Lactococcusnewtdb.jpg , http://www.atcc.org/~/media/Attachments/F/1/2/8/1768.ashx,

HelaT CHO-7 BHK21

pH, temperature, ratio of cell densities, incubation times, glucose concentration