Bacteria Without a Cell Wall L-forms Pros & Cons of Cell Wall - - PowerPoint PPT Presentation

▶
bacteria without a cell wall
SMART_READER_LITE
LIVE PREVIEW

Bacteria Without a Cell Wall L-forms Pros & Cons of Cell Wall - - PowerPoint PPT Presentation

Dec 13, 2022 304 likes •680 views

A New Chassis for Synthetic Biology: Bacteria Without a Cell Wall L-forms Pros & Cons of Cell Wall Cell membrane Cell wall DNA Cell membrane ribosomes RNA metabolites Bacterium with Bacterium cell wall without cell wall Previous

slide-1
SLIDE 1

A New Chassis for Synthetic Biology: Bacteria Without a Cell Wall

L-forms

slide-2
SLIDE 2

Pros & Cons of Cell Wall

Cell membrane DNA ribosomes RNA metabolites

Bacterium without cell wall

Cell wall Cell membrane

Bacterium with cell wall

slide-3
SLIDE 3

Previous work on L-forms

TEM pictures of L-forms

Gilpin, R. W., Young, F. E. & Chatterjee, A. N., 1973. Characterization of a Stable L-form of Bacillus subtilis 168. Journal of Bacteriology, 113(1), pp. 486-499.

  • Discovered by Lister Institute in

1935

  • Roles in diseases such as

sarcoidosis and septicemia

  • Pathogens are not a good

chassis for synthetic biology

  • We engineered the non-pathogen
  • B. subtilis to produce L-forms
  • Built on pioneering work by Prof.

Jeff Errington and colleagues at Newcastle

slide-4
SLIDE 4

Bacillus subtilis

slide-5
SLIDE 5

Aim

To develop L-forms as a chassis for the synthetic biology community

slide-6
SLIDE 6

Synthetic Biology: Engineering Life Cycle

Requirements Design Implementation Verification Maintenance Refinement

Requirements

slide-7
SLIDE 7

Ultimate Goals

  • Develop a switch device that will selectively

turn the cell wall ON and OFF

  • Demonstrate the use of L-forms for real

world applications

slide-8
SLIDE 8

Human Practice & Implications

QUESTION: Are fused cell-wall less bacteria genetically modified? Implications of release of L-forms into the environment

slide-9
SLIDE 9

UK, EU and US Law

slide-10
SLIDE 10

Built-in Kill Switch

L-forms in soil after 1 min incubation

1sec = 1sec

L-forms in normal media NB/MSM

slide-11
SLIDE 11

Synthetic Biology: Engineering Life Cycle

Requirements Design Implementation Verification Maintenance Refinement

Design

slide-12
SLIDE 12

Rule-based Modelling

Standard modelling (eg, SBML) 39 species 184 reactions Rule-based modelling (BioNetGen) 5 molecular types 6 rules

From writer’s perspective

slide-13
SLIDE 13

Model-based Design

slide-14
SLIDE 14

Switch BioBrick:

Modelling Informs Design

Molecule numbers

Molecule numbers

Peptidoglycan biosynthesis in the absence of xylose

Molecule numbers

slide-15
SLIDE 15

Synthetic Biology: Engineering Life Cycle

Requirements Design Implementation Verification Maintenance Refinement

Implementation

slide-16
SLIDE 16

Switch BioBrick:

Implementation

pbpB pbpb spoVD murE murE

Host chromosome BBa_K1185000

slide-17
SLIDE 17

Synthetic Biology: Engineering Life Cycle

Requirements Design Implementation Verification Maintenance Refinement

Verification

slide-18
SLIDE 18

Switch BioBrick: Characterisation

0.8% (w/v) xylose 0.5% (w/v) xylose No xylose 0.5% (w/v) xylose 0.8% (w/v) xylose

slide-19
SLIDE 19

Switch BioBrick in Action

  • B. subtilis rod expressing GFP
  • B. subtilis L-form expressing GFP

1sec = 7hours

slide-20
SLIDE 20

Potential Applications

slide-21
SLIDE 21

Our Applications

slide-22
SLIDE 22

Genome Shuffling

slide-23
SLIDE 23

Genome Shuffling

BBa_K1185001 HBsu-GFP BBa_K1185002 HBsu-RFP

slide-24
SLIDE 24

Implementing Cell Fusion

slide-25
SLIDE 25

L-forms with HBsu-GFP tagged L-forms with Hbsu-RFP tagged

+

Genome Shuffling

L-forms with both HBsu- GFP and RFP tagged L-forms with both HBsu-GFP and RFP tagged

slide-26
SLIDE 26

L-forms and plants

slide-27
SLIDE 27

L-forms Colonise Plants

Brassica pekinensis with Hbsu-GFP tagged L- forms around the cell wall Brassica pekinensis non-innoculated negative control

slide-28
SLIDE 28

Human Practices: Revisited

slide-29
SLIDE 29

Community Interaction

Leeds 2013 iGEM team model using BioNetGen

slide-30
SLIDE 30

Summary

slide-31
SLIDE 31

Our BioBricks

 BBa_K1185000: Enables B. subtilis to switch between a cell walled rod form and cell wall removed L-form, dependent on the presence of xylose in growth media  BBa_K1185001: Non-discriminately tags DNA, allowing location of the DNA by glowing green under fluorescence.  BBa_K1185002: Non-discriminately tags DNA, allowing location of the DNA by glowing red under fluorescence.

slide-32
SLIDE 32

Acknowledgments

  • Dr. Stach
  • Dr. Hallinan
  • Dr. Zuliani
  • Mr. Park
  • Dr. Smith
  • Mr. Gilfellon
  • Ms. Shapiro
  • Dr. Wu
  • Dr. Robertson
  • Prof. Wipat
slide-33
SLIDE 33

Summary

  • A foundational advance: A new chassis for

Synthetic Biology; informed by discussion with ethicists and the public

  • We have created a genetic switch to turn the cell

wall on and off

  • We demonstrated that our engineered L-forms

can be fused to shuffle their genomes

  • We showed that these L-forms can inhabit plants
slide-34
SLIDE 34
slide-35
SLIDE 35

Architecture

Architecture cycle Synthethic Biology cycle