Synthetic auxotrophs for containment

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Assessing and mitigating biosafety risks in
synthetic biology J. Christopher Anderson UC
Berkeley Bioengineering SynBERC

• Synthetic auxotrophs for containment
• Online training modules and certification
• Formal framework for assessing risk

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Synthetic auxotrophy for the containment of
engineered microbes
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Requirements of biosafety strain

• Disenable the transfer of DNAs to and from the
  organism
• rE. coli (codon swapping)?
• Restrict the growth of the organism to the
  application environment
• dapD mutants?
• Unnatural amino acid dependence?
• Self-destruct devices?
• Synthetic auxotrophes?

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Additional requirements

• Modifications cannot be escaped by
  complementation
• Modifications cannot revert through natural
  mutation mechanisms
• Sufficiently inexpensive to allow use in a large
  bioreactor for production of commodity chemicals
• No adverse impact on the stability, metabolic
  load, growth rate, or gene expression of the
  organism
• Be extensible to industrially-relevant organisms

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Synthetic Auxotrophs
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ToxR One-Hybrid System
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Training modules for assessing biosafety risks
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Biological Use Authorization
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Biological Use Authorization
Section VI - Scope of Work Narrative Describe
in lay terms the goal/purpose of experiments,
methods and equipment used in the experimental
procedures. Include safety/containment
procedures, as well as decontamination and
disinfection processes applied during the conduct
of research. In particular, two areas must be
addressed- indicate if you will be autoclaving
your agent/recombinant DNA waste and describe
your spill clean-up procedures. Narrative must
address the potential sources of risk to
personnel (e.g., aerosol generation) and/or the
environment (e.g. pollen or interbreeding with
wild species) and how these risks will be
managed. Indicate if over 10 liters of culture
shall be generated or if agents shall be
concentrated. Explain any boxes checked in
Section V. Limit the narrative to one page (page
4 of the BUA Form). Are there any risks
associated with dual use?
How do you assess these risks?
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Assessment of Risk

• Factors considered in determining the level of
  containment include the following information
  regarding the agent itself
• Virulence/pathogenicity/infectious dose
• Environmental stability
• Route of spread, communicability
• Quantity/concentration/volume used
• Vaccine/Treatment availability
• Allergenicity
• Potential for nefarious dual use

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Training Module for Biosafety
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Training Module for Biosafety

• Does your system include any components from a
  risk group 2() organism?
• Are any of those components known virulence
  factors?
• Does your system contain any components
  associated with select agents?
• Would you expect your agent to cause risk to
  human, animal, or plant health?
• Would you expect your organism to persist in the
  environment?

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Formal assessment of biosafety risks
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Why formalize?

• Software-derived inference mitigates the risk of
  lazy or incomplete subjective assessments
• When systems become more complex, it will be
  harder to assess risk subjectively
• If you can teach a computer to do things
  automatically and reach the correct answer for
  all cases, then you have found a complete formal
  theory
• Analyzing what things do is often easier when you
  approach it from the perspective of a formalism
• In the context of risk, it provides a concrete
  justification for the assessment and a common
  vocabulary for discourse

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First Attempt
Ben Bubenheim

• What is the highest risk group of any specific
  component of the system? (all features and the
  chassis)
• Conventional wisdom is to start with the highest
  risk group and work downward
• Presence of select agent is usually deterministic

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Payload Delivery Device
Jin Huh
Source organism risk group
2
1
1
1
1
1
1
2
1
2
1
1
1
1
1
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Assessing Risk of Devices The payload delivery
device
tnp
gfp
tn
tn
Payload Delivery Device
Peuk
Peuk
Source organism risk group
1
1
1
1
1
1
1
1
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Elements of risk

• The chassis itself can be dangerous
• Any individual component could be dangerous by
  virtue of its molecular function the context of
  a dangerous component is irrelevant
• A device could be more dangerous then the sum of
  its components, independent of the chassis
• In the context of certain chassis, a particular
  device could become dangerous

i.e. Shigella flexneri strain 2457T
i.e. ricin orf and all its encodings
i.e. maitotoxin biosynthetic pathway
i.e. ToxR in Vibrio cholerae
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Elements of risk

• You must include natural mechanisms of evolution
  in the assessment of risk for a given Strain
  (chassis sequence modifications)
• You must include any potential risks of dual use
  of the component Features or compositions thereof
  enabled by the availability of the DNAs
  introduced
• so, ultimately, need a framework that describes
  risks based on compositions of Features (
  Devices) provided a specific Strain

Therefore, cis relationships are irrelevant
Therefore, only essential components should be
part of the description of the dangerous
composition
What exactly is a Device?
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Act Basics
Saurabh Srivastava, Jonathan Kotker, Ras
Bodik, Sanjeet Seshia EECS, UCB
Its (super) Family
Ptet_R0040
A Feature
tccctatcagtgatagagattgacatccctatcagtgatagagatactga
gcac
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Act Basics
Its (super) Family
Ptet_R0040
A Feature
tccctatcagtgatagagattgacatccctatcagtgatagagatactga
gcac
s70 and transcription
TetR
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Act Basics an autorepression device
LacI
Cro
lacI
P_tac
cro
P_RM
TetR
tetR
Ptet_R0040
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Act Basics an autorepression device
trans
cis
X
x
Px
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So what is a device?
A graph wherein the nodes are families and the
edges are cis or trans relationships between the
families

• Devices can exist at multiple levels of
  abstraction based on how specific a family is
  chosen for each node
• Particular parts instantiate a device if features
  belong to the devices families and their
  inferred cis and trans relationships are a
  subgraph of the device

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What does Act give you?

• Provided a Strain composition, it enables
  inference of the devices that resulted from your
  modifications
• It can describe abstractions of the device that
  address substitutions of features, point mutants,
  etc.
• Provided a list of devices of interest it can
  tell you whether a strain contains any of those
  devices

Whats missing?

• The mapping of dangerous cellular function to
  devices
• Enumeration and classification of expected
  dangerous cellular functions

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Some dangerous things

• A yeast cell expressing the active form of prion
• A lab strain of E. coli with various
  modifications throughout its genome to enable
  oral to intestine colonization that
  constitutively expresses ricin
• A probiotic strain of E. coli that has been
  transformed with a morphine biosynthetic device.
• An engineered Salmonella strain with a
  cell-surface displayed peanut eptitope