Microbes in Synthetic Biology 2: Directed Evolution

Learning Outcomes

• Understand how mutation and selection form the basis of evolution.

• Be able to apply this insight into methods for directed evolution.

• Understand how we can control the rate of mutation experimentally.

• Understand methods by which we can select and screen for particular mutations that we want.

Intro

• Evolution:

  • Change in the heritable characteristics of biological populations over successive generations.

  • Heritable

  • Population

  • Generations = long time.

Motility in Life

• Many organisms can move.

• Exploration of new hosts, habitats, and niches.

• Flagellar motility is a crucial virulence factor.

• Motility in many bacterial and archaeal species involves an organelle called the flagella.

• Movement of bacteria occurs via controlling how often you are in a ‘run’ state (swimming forward) and how often you are in a ‘tumble’ state, re-orienting.

The bacterial flagellar motor: brilliant evolution or intelligent design?

• The complexity of nature's most impressive swimmer leads some to mistakenly believe it was designed, but it is proof of evolution at work.

• Flagella-powered bacteria swim hundreds of body lengths in a second and can change direction in a fraction of that time.

• The bacterial flagellar motor rotates up to five times faster than a Formula 1 engine.

• The motor builds itself by assembling its constituent parts.

• The flagellar motor is one millionth of the size of a grain of sand.

Flagellum

• Bacterial Flagellar Motor Vs Injectisome

• The flagellum and injectisome contain proteins that are highly structurally and functionally related.

• The C ring, the MS ring, and the apparatus that exports proteins outside the cell wall contain nine proteins that evolved from common ancestors.

Evolution or intelligent design? The story of the bacterial flagellar motor

• Bacteria with flagellar motor

What is Evolution?

• Directed evolution:

  • A method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids toward a user-defined goal.

• Experimental evolution:

  • The use of laboratory or controlled field manipulations to investigate evolutionary processes.

  • Designing experiments to study the molecular processes of evolution.

What is the basis of evolution?

• Evolution is really about two things:

  • Mutation

  • Selection

• “In microbiology, the roles of mutation and selection in evolution are coming to be better understood through the use of bacterial cultures of mutant strains.” - Edward Tatum, Nobel Laureate Medicine 1958.

Nobel Prize for Chemistry in 2018

• Half to Frances Arnold for:

  • “the directed evolution of enzymes”

• The other half to George Smith and Gregory Winter for:

  • “the phage display of peptides and antibodies”

Nobel Prize for Medicine in 2022

• Discoveries concerning the genomes of extinct hominins and human evolution.

• Svante Pääbo, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany; Okinawa Institute of Science and Technology, Okinawa, Japan

Paleogenomics

• In modern day humans with European or Asian descent, approximately 1-4% of the genome originates from the Neanderthals

Nobel Prize for Chemistry in 2018

• Half to Frances Arnold for:

  • “the directed evolution of enzymes”

• The other half to George Smith and Gregory Winter for:

  • “the phage display of peptides and antibodies”

Directed Evolution Illustration

• Random mutations are introduced in the gene for the enzyme that will be changed.

• The genes are inserted in bacteria, which use them as templates and produce randomly mutated enzymes.

• The changed enzymes are tested. Those that are most efficient at catalyzing the desired chemical reaction are selected.

• New random mutations are introduced in the genes for the selected enzymes. The cycle begins again.

Arnold Classics

• Arnold, F.H. (1998). Design by Directed Evolution. Acc. Chem. Res. 31, 125–131.

  • “The chemist is not terribly impressed with a synthesis requiring a reactor the size of a football field simply because the enzyme functions in water and the substrate does not dissolve”

  • “Irrational design”

Matt Baker on Frances Arnold

• Enthusiasm for Frances Arnold's keynote at Lorne Proteins conference.

• Directed evolution of microbial enzyme opens fresh route to carbon-carbon bond formation.

• @francesarnold, Nobel Laurate

Phage Display

• Presents genetic information outside a cell as a peptide that we can then use for screening.

• This saves a lot of time and means we can interrogate the function or binding of a peptide on a lot of cells at once.

Antibody Display and Antibodies

• Antibody structure and function recruits effector functions.

• IgG mAbs are large (150,000 Da) Y-shaped protein molecules with two (H/L) chains.

• Associated VH/VL domains (=Fv at end of Fab arms) come together to form antigen binding site comprising a scaffold with six loops of variable sequence.

• Variability created by combinations of multiple genetic segments.

• Ab binds to infectious agent and can block infection, also can kill infectious agent by recruiting effector functions through Fc domains (stem).

Massive Commercial Benefit

• “antibody display provided a set of turnkey processes that ultimately fueled biopharmaceutical pipelines”

• In 2018, around 60% of all therapies in development for oncology were antibody based.

• Nobel work that galvanized an industry; Nature Biotechnology volume 36, page 1023 (2018)

• 8 years: Using Arnold's approach, the Novo Nordisk group created a bleach-stable heme peroxidase variant with 174 times the thermal stability and 100 times the oxidative stability of the wild-type enzyme.

Phage Display Illustration

• The genetic information for the antibody's binding site is inserted into the phage's DNA. After this, a library with a huge variety of antibodies is created.

• The phage with strong attachments to a specific target are selected.

• Before another selection is conducted, random mutations are introduced into the antibodies that attached to the target.

• With each subsequent generation, the antibodies attach more strongly and with increasing specificity to the target protein.

Methods to Introduce Mutations?

• How to introduce mutations?

  • Controlled mutagenesis

  • Buy things scrambled (NNS)

  • Use mutator strains.

• How to select?

  • Manually

  • Semi-manually (robotics/plates)

  • Automatically

Error Prone PCR

• Simple

• Can get up to 10% mutation rate per position

• Can tune the ‘amount of mutation’

• Basis:

  • Take Taq and ‘make it bad’

  • Naturally $1 \times 10^{-4}$ error rate

  • We want around 1% at nucleotide level

  • Which is around 2% at amino acid level

  • ~30% mutations silent

How to make Taq ‘bad’

• Increase $Mg^{2+}$

  • Stabilizes non-complementary pairs, i.e., stabilizes formed errors

• Add $Mn^{2+}$

  • Reduces the specificity, i.e., allows errors to form more easily

• Unbalanced dNTP concentration

  • Force misincorporation, and rebalance non-equivalent effect of $Mn^{2+}$

Controlled Mutagenesis

• Mutagenic PCR

• Error-Prone PCR

Mutators

• XL1-Red (Stratagene)

  • Mutations in their DNA repair pathways

  • Results in 5000-fold increase in spontaneous mutations.

  • $3.5 \times 10^{-6}$ vs $7 \times 10^{-7}$

  • Problem is mutations are everywhere.

XL1-Red

• XL1-Red is deficient in three of the primary DNA repair pathways.

  • The mutS (error-prone mismatch repair),

  • mutD (deficient in 3´ - to 5´ - exonuclease of DNA polymerase III)

  • mutT (unable to hydrolyze 8-oxodGTP).

• Consequences:

  • Doubling time 90-120 mins (!)

  • The high mutation rate of the XL1-Red mutator strain causes a wide range of colony sizes.

  • XL1-Red mutator strain should not be propagated on plates for prolonged periods of time, because it can get whacky.

Buy DNA with every combination

• Expensive:

  • B-unit stator, MotB, say I want to scramble 70 residues ~ 200bp:

  • $200 \text{ bp } \times 70 \text{EUR}$

Screening

• Adding mutations is relatively easy.

  • Although controlling it is less so.

• “the primary limitation in most directed evolution experiments is the identification of the desired mutants.”

  • Plates

  • Display

  • Sorting

Plates

• Old faithful, 96 well plate

• Robotics helps massively

• Can get crazy if you want to (9600 well plate)

• Colorimetric or fluorometric assays.

• Can shake and grow

Using the cell

• Cell surface displays:

  • Peptides and proteins can be displayed on the surface of microbial cells by fusing them with the anchoring motifs.

Sorting individual cells

• If the cell is colored/fluorescent, can use fluorescent activated cell sorting (FACS)

High throughput selection

• Screening typically means something is doing it, making a decision.

• Selection means we build it so the biological system does it itself.

• Much larger library, e.g., $10^{11}$ combinations.

• Plate is maybe $10^5$

Experimental Evolution in action

• Scientists reveal the frightening speed at which bacteria can develop antibiotic resistance

Lenski Experiment

• Long-term evolution experiment

Directed Evolution of Motility

Learning Outcomes

• Evolution requires mutation and selection. To direct evolution, for a purpose, we need to control both.

• Mutation:

  • Error-prone PCR (EP-PCR)

  • Mutator strains

• Screening:

  • Plates or manually

  • Robots

  • Sorting

• Selection:

  • Engineer biology to do the selection: Link protein function to growth or death.

Practice Quiz Questions

• What two things are required for the process of evolution to occur?

• What is one way to introduce mutations to a specific gene of interest?

• To make PCR error prone, what can be done to increase the mutation rate?

• What is phage display and where can it be useful?