21 st Century Antibiotics Gram Negative Antibiotic Gram Positive - - PowerPoint PPT Presentation

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21 st Century Antibiotics Gram Negative Antibiotic Gram Positive - - PowerPoint PPT Presentation

Feb 06, 2024 26 likes •449 views

21 st Century Antibiotics Gram Negative Antibiotic Gram Positive Antibiotic Plasmid Library Software 21 st Century Antibiotics Gram Negative Antibiotic Gram Positive Antibiotic Plasmid Library Software We can engineer better Gram-negative

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21st Century Antibiotics

Gram Negative Antibiotic Gram Positive Antibiotic Plasmid Library Software

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21st Century Antibiotics

Gram Negative Antibiotic Gram Positive Antibiotic Plasmid Library Software

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We can engineer better Gram-negative antibiotics using synthetic biology

  • wipe out normal gut flora
  • taken after symptoms

are obvious Problems with traditional antibiotics:

  • widespread resistance

Engineered probiotic

  • works before symptoms
  • activated when pathogen

is detected

  • specific for Gram-

negative organisms

  • novel therapeutic
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Probiotic Detects Pathogen

toxin anti-toxin

Probiotic Expresses Toxin and Antitoxin Probiotic Secretes Toxin

Engineered probiotic detects and destroys Gram-negative pathogens

toxin anti-toxin toxin anti-toxin

Essential Probiotic Components

  • Cell chassis
  • Protein secretion system
  • Inducible toxin/antitoxin system

X

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The Type VI Secretion System punctures the cell wall

  • f target Gram-negative cells and injects proteins

T6SS bacteria cytoplasm target bacteria cytoplasm

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The Type VI Secretion System punctures the cell wall

  • f target Gram-negative cells and injects proteins

T6SS bacteria cytoplasm target bacteria cytoplasm

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Fosmid containing T6SS does not express in E. coli

50kb Type VI Secretion Fosmid Promoter Region

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Engineered fosmid directs expression of the T6SS in E. coli

Uninduced Induced Type VI Protein

  • P. aeruginosa
  • E. coli
  • E. coli

Type VI Protein in E. coli Positive Control protein

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Tse2Tsi2 is a toxin/antitoxin system recognized by the T6SS

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Toxin/Antitoxin is induced in response to an example bacterially-produced small molecule

Image created via TinkerCell BioBrick F2620 Toxin/Antitoxin Operon

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Toxin/Antitoxin is induced in response to an example bacterially-produced small molecule

BioBrick F2620 Toxin/Antitoxin Operon

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Probiotic Detects Pathogen

toxin anti-toxin

Probiotic Expresses Toxin and Antitoxin Probiotic Secretes Toxin

Engineered probiotic detects and destroys Gram-negative pathogens

toxin anti-toxin toxin anti-toxin

Essential Probiotic Components Expressed Type VI secretion in E. coli, fosmid available Characterized, BioBricked and submitted Tse2/Tsi2 X

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21st Century Antibiotics

Plasmid Library Software Gram Negative Antibiotic

Characterized, BioBricked, and submitted Tse2/Tsi2 Submitted Fosmid containing T6SS Expressed Type VI secretion in E. Coli

Gram Positive Antibiotic

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No Anthrax (live or dead) was used in this project!

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Removing Bacillus anthracis protective coat makes it vulnerable to immune system

Protected Anthrax Vulnerable Anthrax Decapsulation

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Redesign CapD to break down protective coat

Transpeptidation (Native) Hydrolysis (Desired)

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CapD auto-cleaves to reveal active site

CapD : Key Catalytic Residue

* *

N C Inactive CapD

*

C’ Active CapD N’ N C

Self Cleavage

*

Inactive Enzyme Active Enzyme Active Enzyme

*

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Avoiding auto-cleavage: redesigning CapD to produce enzyme in active state CapDCP

Circular Permutation

CapDCP Active CapD_CP C *N

: Key Catalytic Residue

*

N C Inactive CapD

*

Genetically encoded peptide linker

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CapDCP is easy to express and quantify!

Active Enzyme

CapD CapDCP

*

: Key Catalytic Residue

*

Inactive Enzyme Active Enzyme Active Enzyme

*

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CapDCP shows enzymatic activity!

: Key Catalytic Residue

*

kcat (hr-1) Km (nM) kcat (hr-1) Km (nM) CapD 27.2 ± 1.5 22.1 ± 2.9 1.8 ± 0.3 2.4 ± 1.4 CapD_CP 67.2 ± 7.5 23.2 ± 5.4 4.0 ± 0.1 0.6 ± 0.1 Transpeptidation Hydrolysis

Active Enzyme Inactive Enzyme Active Enzyme Active Enzyme

CapD CapDCP

* *

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Using FoldIt to design a better anthrax destroyer

FoldIt: Developed by the Baker and Popović Labs at the University of Washington

Mutate

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F24H, R356K T2V T20S T59N F24Y L40R S143K CapDCPNFDelta L40S L40W M61S N23K T20C L40A T2S T20S,T59S_M61S F24H F24H, L40R, T59N_M61S T20S, L40D T20S, L40F T20S, L40R, N81Q CapD

0.2 0.4 0.6 0.8 1 1.2 1.4 0.2 0.4 0.6 0.8 1 1.2 1.4

Transpeptidation Relative to CapD_CP Hydrolysis Relative to CapD_CP CapD_CP

Improved Variant

Designed, built, & tested 87 variants Succeeded in altering the reaction specificity

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Michaelis-Menten profile shows 10 fold switch in reaction specificity

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21st Century Antibiotics

Plasmid Library Software Gram Negative Antibiotic

Characterized, BioBricked, and submitted Tse2/Tsi2 Submitted Fosmid containing T6SS Expressed Type VI secretion in E. Coli

Gram Positive Antibiotic

Created and characterized 87 CapD_CP mutants BioBricked and submitted CapD, CapD_CP, and best variant Collaboration to test best variant on live Anthrax c

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21St Century Antibiotics

Plasmid Library Software Gram Negative Antibiotic

Characterized, BioBricked, and submitted Tse2/Tsi2 Submitted Fosmid containing T6SS Expressed Type VI secretion in E. Coli

Gram Positive Antibiotic

Created and characterized 87 CapD_CP mutants BioBricked and submitted CapD, CapD_CP, and best variant Collaboration to test best variant on live Anthrax

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Protein Expression Vectors

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Protein Expression Vectors

pSB3K3 pSB3K3 + f1 origin 3kb 2 kb 1.5 kb

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psb1A3 expression cassettes with gfp

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WikiDust makes interactive diagrams that link directly to the parts registry

WikiDust

TinkerCell developed by the Sauro Lab at the University

  • f Washington
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PartsRobot

PartsRobot simplifies the BioBrick submission process

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21st Century Antibiotics

Plasmid Library

3 New BioBricks (F1 origin, new LacI, new T7 promoter) 4 New BioBrick protein expression cassettes Characterized existing Registry promoters

Software

WikiDust Allows users to quickly generate diagrams that PartsRobot simplifies registry submission process link to the parts registry – BBF RFC 68

Gram Negative Antibiotic

Characterized, BioBricked, and submitted Tse2/Tsi2 Submitted Fosmid containing T6SS Expressed Type VI secretion in E. Coli

Gram Positive Antibiotic

Created and characterized all CapD_CP mutants BioBricked and submitted CapD, CapD_CP, and best variant Collaboration to test best variant on live Anthrax

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Faculty

  • David Baker
  • Joseph Mougous
  • Herbert M Sauro
  • Eric Klavins

Advisors

  • Ingrid Swanson
  • Michal Galdzicki
  • Justin Siegel
  • Josh Bishop
  • Matthew Smith
  • Rob Egbert
  • Jeremy Mills
  • Deepak Chandran
  • Joe Harrison

Space Donations

  • Alan Weiner
  • Dominic Chung
  • Ling Liu

UW Microbiology Department UW Biochemistry Department UW MCB Department Arthur Friedlander at USAMRIID Michael Jacobs at UW

Acknowledgements from the 2010 UW iGEM Undergrads

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Questions?

Plasmid Library

3 New BioBricks (F1 origin, new LacI, new T7 promoter) 4 New BioBrick protein expression cassettes Characterized existing Registry promoters

Software

WikiDust Allows users to quickly generate diagrams that PartsRobot simplifies registry submission process link to the parts registry – BBF RFC 68

Gram Negative Antibiotic

Characterized, BioBricked, and submitted Tse2/Tsi2 Submitted Fosmid containing T6SS Expressed Type VI secretion in E. Coli

Gram Positive Antibiotic

Created and characterized all CapD_CP mutants BioBricked and submitted CapD, CapD_CP, and best variant Collaboration to test best variant on live Anthrax

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F F PDG Linker

Catalytic knockout confirms active site chemistry

Q Q

Assay utilizes synthetic fluorescent peptide

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Expected weight of CapD_CP without Methionine=55285Da, with Methionine=55417Da. Our mass spec detected a peak at 55274.8Da (no Methionine) well within the 0.02% error limit for our mass spec

Mass Spec Verifies Start Methionine Removed

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Circular Permutation

Linker ~526 1 1 351 352 526

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Notes

  • Pixilate PacMan
  • Switch focus to removing tarnspeptidation not

increasing hydrolosys

  • Get new protein expression efficiency
  • Change anthrax slide to get rid of mariners

and Yankees

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Tse2 toxicity