Optimization of Biofilm Formation

for Use in Water Filtration Systems Purdue University iGEM

Team 2012

Water: A Grand Challenge

• Over 1 billion people do not

have adequate water access

• Major health concerns

caused by lack of clean water

• Need a solution

• Sustainable

• Inexpensive Source: US AID

Bioreactors for Water Treatment

• Advantages

• Organic contaminant

• Inexpensive inputs

• Disadvantages

• Constantly Mixed

• Large amount of space

= Organic Contamination = bacteria

Clean, Filtered Water

http://hugroup.cems.umn.edu/Cell_Technology/Notes/Cell%20Culture%20Bioreactors.pdf

Biofilms in Bioreactors

= Organic Contamination = bacteria

Clean, Filtered Water

• Water passes over

biofilm

• Reduces space needed

• Eliminates need for mixing

• Works as a biofilter

Van Loosdrecht, M.C.M., Heijnen, S.J. Biofilm bioreactors for waste water treatment

What are Bioflims?

• Community of one or

more types of bacteria

• Bacteria and EPS

• Increased Resistance

• Adhesion to surfaces

• Abiotic

• Biotic

• Currently used as

biofilters

Zhang, W. Characterization of passive and active biofilm detachment

Our Solution: Optimizing Biofilms

• Goal: optimize cellular

resources to create biofilm

to act as a biomechanical

filtration system

• Biomechanical Filter:

• Biological Filter: E. coli bacteria

rapidly form a biofilm that

adheres to a surface within the

bioreactor

• Mechanical Filter: Silica binding

peptides are produced to create

a silica matrix

Picture of the membrane

aerated bioreactor we

used in lab

Not to scale

Addition of Curli Protein to outer membrane allows

for rapid formation of biofilms

= curli protein

Not to scale

Enhancing Cellular Adhesion

• Need an adhesion protein that

promotes, thick, fast-forming

biofilms. Our pick: Curli

• Properties

• Natural Bacterial Protein

• Amyloid Fiber

• Adheres to a variety of surface

materials and other cells

• Major extracellular protein in

biofilms

(-) Curli

(+) Curli

Vidal, O., & Longin, R. (1998). Isolation of an Escherichia coli K-12 Mutant Strain Able To Form Biofilms on Inert

Surfaces: Involvement of a New ompR Allele That Increases Curli Expression. J Bacteriol,180(9), 2442–2449.

Retrieved June 12, 2012, from http://www.ncbi.nlm.nih.gov/pmc/articles/

Adhesion Construct

• OmpR234: Mutant regulatory

protein

• Constitutively upregulates Curli

cluster expression within

bacteria

pTet RBS OmpR234 Term

• Part BBa_K932000

OmpR234

Curli Operon

EnvZ

Curli Filament

Silica Binding Construct

• Designed a fusion peptide to bind silica monomers on the cell surface

• Outer Membrane Protein A (OmpA)

• Silicatein Alpha (Silα)

pLac RBS OmpA-Silα TetR Term

Curnow, P. (2005). Enzymatic Synthesis of Layered Titanium Phosphates at LowTemperature and

Neutral pH by Cell-Surface Display ofSilicatein-r. JACS, 127, 15749-15755.

Biosilicification: A Mechanical Filter

Treatment with SiO2

Membrane Integration

Silica Matrix Formation

• E. coli expressing OmpA-Silα will bind SiO2 to their surface

• The resulting silica matrix will act as the additional filter in the bioreactor

Silicon dioxide-

OmpA Domain-

Silα Domain-

Induction and Inhibition

TetR

IPTG

Experimental Design Compare

Expression of Curli:

Growth of biofilm with Curli

Static biofilms

Biofilms in bioreactor

Detachment of biofilm with silica

Over-Expression of Curli Filaments

Goal: quantifiably exhibit the presence of curli filaments on the

outer membrane of our transformed cells

• Incubate E. coli cells in

Thioflavin T solution

•Thioflavin T binds to curli

•Excitation and emission

spectrum shifts

•Measure the emission

spectrum and compare to

control

Procedure:

0

100

200

300

400

500

600

Control BBa_K932000

Flu

ore

sce

nce

Un

its

Experimental Group

Thioflavin T Fluorescence Test

for Curli Expression

***

www.wetware.org

Growth Curve of E. coli

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 200 400 600 800

OD

60

0

Time (min)

Control

Bba_K932000

www.wetware.org

Goal: determine if the excess expression of curli filaments taxes

the cell and decreases the rate of cell growth

• Grow the cells to OD of 1

• Transfer given amount of cells

to SOB and culture in 370 C

while shaking

• Record the OD every hour or

half hour depending on the

doubling time

Procedure:

Static Biofilms Goal: quantify the amount of cells attached to an abiotic surface

at given time intervals

Example of the biofilm

stained with crystal violet

• Grow media + bacteria in 24 well plates

• After given time wash out excess media

and bacteria

• Stain remaining biofilm with crystal violet

• Measure the OD of the biofilms to

determine the cell density

Procedure:

www.wetware.org

Curli Enhances Biofilm Formation Rate

0

0.5

1

1.5

2

10^6 2*10^6 3*10^6Ab

so

rba

nce

Un

its (

AU

)

Starting CFU/mL

Static Biofilm Formation (24 hrs)

Control BBa_K932000

0

0.5

1

1.5

2

10^6 2*10^6 3*10^6Ab

so

rba

nce

Un

its (

AU

)

Starting CFU/mL

Static Biofilm Formation (48 hrs)

Control Bba_K932000

0

0.5

1

1.5

2

2.5

10^6 2*10^6 3*10^6Ab

so

rba

nce

Un

its (

AU

)

Starting CFU/mL

Static Biofilm Formation (72 hrs)

Control Bba_K932000

Biofilms in Bioreactor

Rt: membrane aerated bioreactor

without media

Lt: Media in MABR

Goal: prove that our bacteria can grow in a similar

environment to what is found at an industrial level

•Introduce cells and media into

bioreactor

•Feed bioreactor with minimal media

every three days

•Clean bioreactor with PBS every third

cleaning

Procedure:

www.wetware.org

Results from Bioreactor Biofilms

Biofilm of E. coli + curli after

30 days in MABR

•Biofilms grew for 15 and 30 days

•Systems: •E.coli 15 days

•E. coli 30 days

•E. coli + silica 15 days

•E. coli + silica 30 days

•Curli 15 days

•Curli 30 days

•Inadequate biofilms due to time

restraints

•Visible biofilms for Curli 30 days

•None of the rest were visible

without microscope www.wetware.org

Detachment of Cells from Biofilm

Goal: quantify the amount of bacteria and bacterial

aggregations that detach from biofilms

• Subject biofilms to flows

of PBS

•Collect samples

•Use Coulter Counter to

determine the number

and sizes of particles

Procedure:

How we held the biofilms in place

www.wetware.org

Detachment Experimental Setup Flow of water

Flow of water

Control: E. coli

Experiment: E. coli + Curli

Bacteria and water

is collected and

analyzed with

Coulter Counter

www.wetware.org

Curli Expression

- + B

iosilic

ific

ati

on

-

+

Quantitative results

Wei

ght%

0

20

40

60

80

100

C O Si Pt

Curli Expression

- +

Bio

silic

ific

ati

on

-

+

Elemental Analysis of Biosilifcation

Quantitative results

Wei

ght%

0

10

20

30

40

50

60

C O Si Pt

Quantitative results

Wei

ght%

0

20

40

60

80

C O Si Pt

Quantitative results

Wei

ght%

0

10

20

30

40

50

C O Si Pt

Future Work

Characterize detachment with silica binding

protein

Integrate into industry

Test bacterial strains used in industry

Test on a heterogeneous community of cells

Generation STEM: What Girls Say about Science, Technology, Engineering, and Math, http://www.girlscouts.org/research/pdf/generation_stem_full_report.pdf Knewton Infographic : STEM Education, http://columnfivemedia.com/work-items/knewton-infographic-stem-education/

Biomaker Bench Harlem DNA

Labs GenSpace BioCurious

Location Noblesville, IN Harlem, NY Brooklyn, NY Silicon Valley, CA

Demographic K-12 Students &

Teachers

K-12 Students

& Teachers

Retirees

Artists

Youth

Young Professionals

Funding

Institutional &

Corporate

Sponsors

Institutional

Funding

Membership

Fees Membership Fees

Staffing

Student and

Community

Volunteers

Paid full time

faculty

Part Time

Faculty Full time Faculty

Specialty Education and

Outreach Education

Bio-Art

Education Entrepreneurship

After-School Program (Mentoring)

High School level iGEM Teams

Open Community Workshops

Teacher Certification Workshops

State-wide field trips

Business incubator space

•After-School Programming

•High School iGEM

•Community Workshops

•Teacher Certification

•Entrepreneurship Classes

•Idea Incubation Space

•Local Outreach

Scaffold of Use

Start:

Identify

Problems

and

Solutions

• Primary

and

Secondary

Research

• Assess Feasibility

• Establish

partnerships

• Present Plan

and consult

Noblesville, IN • Secure

Funding and

Resource

Allocation

• Establish

Space

Start:

Commissione

d by GS

• Vision Statement

• Curriculum

Development

• Local Trials

• Reevaluation

• Propose to

Nationals

• National

Implementation

Acknowledgements

Our Faculty Advisors:

Drs. Jenna Rickus and Dr. Kari Clase Graduate and Post Doc Students:

Jing Lu

Janie Brennan

Soo Ha

Rajtarun Madangopal

Tony Pedley

Rouxi Wu

Facility: Bindley Bioscience Center

The Team

Purdue iGEM Team:

Sean Kearney – President

Peter Mercado-Reyes – Treasurer

Max Showalter – Secretary

August Clevenger

Rachel Feltner

Mrudula Vemuri

Namita Balchander

Haefa Mansour

James Nolan

Chris Thompson