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"Synthetic biology: containment and release of engineered micro-organisms" “Horizontal gene transfer in the environment” Pascal SIMONET Environmental Microbial Genomics Group, Laboratoire AMPERE UMR CNRS 5005 Ecole Centrale de Lyon In response to: “Building-­‐in Biosafety for SyntheJc Biology” Oliver Wright, Guy-­‐Bart Stan and Tom Ellis Microbiology 2013 (in Press. Published March 21, as doi:10.1099/mic.0.066308-­‐0) « Preparing syntheJc biology for the world » Gerd H.G.Moe-­‐Behrens, Rene Davis and Karmella A. Haynes Front. Microbiol. January2013|Volume4|Ar]cle5 | 1 The majority of research and development in synthe]c biology has u]lised microbes as the host cell Superbug? Could gene]cally modified microbes (GMMs) outcompete na]ve species and disrupt habitats? Synthe]c gene(s) or operon(s) HGT ? Superbugs? At the 1975 Asilomar conference, scientists agreed on a cautious approach, incorporating both physical and biological containment.
use of “syn-­‐genic” material, i.e. novel sequences not found in nature? (Wright, Stan, Ellis 2013)
Superbugs? The « wrong » quesJon ! “Laboratory acclima]sed cells are largely unable to establish themselves in the environment in a meaningful way and face a range of serious efficacy issues even during short-­‐term reten]on by a habitat” (Wright, Stan, Ellis 2013). • Strong physical, chemical and biological heterogeneity of natural environments. • Huge natural microbial diversity and strong compe]]on. • No natural “superbugs”. Bacteria more or less adapted to specific condi]ons. “Extreme environments” presence of a significant microbial diversity. The right quesJon: « How to create Efficient and CompeJJve bugs? »
“As most engineered cells are being made to perform work superfluous to their cri]cal func]ons, they are likely to be out-­‐competed beyond the comforts of the lab and therefore either die out or eject their synthe]c circuits” (Wright, Stan, Ellis 2013). HGT (in nature) Cri]cal mechanisms for adapta]on and evolu]on (Complete Genome Sequences, ARG dissemina]on). Referenced mechanisms : Conjuga]on, transduc]on, natural transforma]on. Addi]onal mechanisms: Passive (chemically or physically mediated-­‐) transforma]on. In situ Regula]on: 3 barriers (physical, physiological, gene]c). Importance (and frequency) of HGT (in nature) (example 1) An]bio]c producing microorganisms: The structure of polykeJdes produced by PKSI is related to the specific linear organizaJon in domains and modules. Domains and Modules are naturally transferred among bacteria. Comparison of physico-­‐chemical and structural proper]es of compounds issued from natural sources versus chemical molecules has revealed that the chemical diversity generated by random chemistry is far from the variety of natural ac]ve scaffolds. Importance (and frequency) of HGT (in nature) (example 2) Bacterial degradaJon of xenobioJc compounds. γ-Hexachlorocyclohexane: lindane Organochloride pes]cide synthesized from 1946. No similar compound found in nature. Non polluted soils: Cul]va]on : No degrada]ve-­‐lindane bacteria. PCR : No Amplifica]on Metagenomic DNA analysis: Gene]c and phenotypic screening : Nega]ve Highly polluted soils: RouJne detecJon (and isolaJon) of degradaJve bacteria. Sphingobium francense The gene linA is involved in the first and essen]al step of the aerobic bacterial metabolic pathway in Sphingobium francense and other isolated degrading bacteria. Origin of the linA gene ? linA
linB
linC
linD
linE
linF
A. Mutated gene MutaJon of an ancestor linA-­‐like gene leading to a new acJvity. Search for ancestor genes in non-­‐polluted soils B. Mosaic gene Assembling of a new gene by HGT-­‐based acquisiJon of DNA fragments and recombinaJon. Search for corresponding DNA fragments and HGT and recombinaJon mechanisms Identify linA fragments in different soil environments
(polluted and non-polluted)
1. Bio-informatic analysis of linA (MEME*, NCBI BLAST)
2. Cut in 4 regions
I
3. Design specific
primer sets
4. Application of primers to
soil extracted DNA
II
III
IV
DNA extracted
from soil
Non polluted soil
Cloning
Sequencing
400
300
200
100
*Multiple Expectation Maximum for Motif Elucidation
POSITIVE
CONTROL:
210
polluted soil
Experimental approach:
DNA shuffling
DNA
pairing
synthesis
of homologous
with polymerase
bases
From various DNA fragments
à reassembling
à new DNA molecule
à simulation of a biological process?
DNA
denaturation
fragments
thermocycler
(Boubakri et al., 2006 Gene)
de novo generaJon of linA gene by shuffling promoter
linA
A1-A2
Ar-A6
A9-A11
A7-A8
1. linA fragments or soil
amplified PCR products
2.Genetic
reassembly
A10-A12
A5-Afr
A13-A14
3.PCR
Amplification
4. Cloning in E.coli
5. Screening on medium containing lindane
6. lindane degradation capacity ?
(0,5g/l)
de novo generaJon of a linA gene in vivo (Sphingobium francense and other bacteria) linA construc]on required: Numerous transfer events in a very short period of ]me : 1. HGT involved for a rapid (but not op]mized) response to new environmental condi]ons. S. francense : Strong instability : Gene lin A carried by an instable plasmid. Gene linA flanked by IS. Frequent rearrangements. Other lin genes spread on other replicons. No operons. 2. Op]miza]on (fine tuning): A very long process involving point muta]ons, endogenous rearrangements, etc. Step 1 or step 2 as the best target for syntheJc biology?? S. francense (step 1 resul]ng) not representa]ve of step 2 resul]ng microorganisms. HGT (in nature) Cri]cal mechanisms for adapta]on and evolu]on (Complete Genome Sequences, ARG dissemina]on). Referenced mechanisms : Conjuga]on, transduc]on, natural transforma]on. Addi]onal mechanisms: Passive (chemically or physically mediated-­‐) transforma]on. In situ RegulaJon: 3 barriers (physical, physiological, geneJc). In situ RegulaJon of HGT • Physical contact between recipient and donor (or their DNA) organisms. • AcJve metabolism of donor (or availability of DNA) and recipient bacteria. « Hot spots » for gene transfers? VISUALIZATION of HGT in PLANTA
HGT between GMP (transplastomic) and soil bacteria. Independent gene transfer events. VISUALIZATION of HGT in PLANTA
HGT between GMP (transplastomic) and soil bacteria. Colony forma]on from clone mul]plica]on. GMP: Inac]va]on of the gene]c barrier between plant and bacteria. HGT: Detectable: Homologous recombina]on between donor DNA (plant transgene) and recipient bacteria genomes. RegulaJon of HGT and gene fixaJon Physical barrier: Strength = low
Persistence of DNA Physiological barrier: Strength = low
Ac]ve bacterial metabolism. Passive transforma]on mechanisms Gene]c barrier: Strength = high* Limited pollu]on of bacterial genomes by eukaryo]c DNA. GMM Strength = decreasing
Depends on sequence similarity level Synthe]c GMM Strength = high
No sequence similarity Fixa]on of the new gene in the bacterial popula]on. Gene]c burden or fitness ac]vator?? * Genome integra]on by illegi]mate recombina]on. Efficiency several orders of magnitude lower than HR. Efficiency of safety mechanisms and geneJc devices Wright, Stan, Ellis 2013, Moe-­‐Behrens, Davis, Haynes 2013 At best: safety mechanisms and gene]c devices : Can reduce the probability of HGT. (without considering orthogonal biological systems and xenobiology: Wright, Stan, Ellis 2013, Moe-­‐Behrens, Davis, Haynes 2013). Mul]ple devices of different types in the same bug? Can increase efficiency but will never offer total guarantee. “The higher the complexity of a safety device, the more prone it may be to disturbance and failure. ‘cost’ of each component, as several in tandem will place an undesirable physiological burden on the host and in turn act as a selec]ve pressure to eject the system” (Wright, Stan, Ellis 2013). HGT: The main adapJve weapon of the best adaptable organisms on this planet. HGT: unavoidable. Impact: Negligible (wild bacteria are too strong!!) The right quesJon: How to create Efficient and CompeJJve bugs?