- E. teamwork - Engineering a synthetic microbial consortium 1 2 - - PowerPoint PPT Presentation

• E. teamwork -

Engineering a synthetic microbial consortium 1

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No stable ratio in mixed cultures

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Stable ratio necessary! 1 : 1 : 1

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• E. teamwork -

Engineering a synthetic microbial consortium

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Regulation of mixed cultures by Quorum Sensing

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Quorum Sensing: Communication in bacterial populations

Inactive transcription factor Inducer Inducer synthase Inducible promoter

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Quorum Sensing: Communication in bacterial populations

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Inactive transcription factor Inducer Inducer synthase Inducible promoter

Switching functional QS units creates dependency

Inactive transcription factor Inducer Inducer synthase Inducible promoter

9 Rhl Lux Las

STO P

Switching functional QS units creates dependency

Inactive transcription factor Inducer Inducer synthase Inducible promoter

9 Rhl Lux Las

Promoter

inducible

Each device consists of 3 operation units

Promoter const.

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Promoter const.

Promoter

inducible

ampR

Promoter const.

Each device consists of 3 operation units

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Promoter

inducible

Promoter

inducible

ampR

Promoter const. Promoter const.

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Each device consists of 3 operation units

ampR

Triangle of dependency

ampR ampR

Inactive transcription factor Inducer Inducer synthase Inducible promoter

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c c

ampR

Bidirectional dependency

Red construct

ampR ampR

Blue construct

Inactive transcription factor Inducer Inducer synthase Inducible promoter

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The BioBrick construct

Blue construct: Red construct:

• 15 BioBricks
• 2 final constructs
• 24 new standard devices for Registry

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ampR

Operation Unit 1: Reporters

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• E. coli cells in monoculture express the

chromoproteins eforRed (left) and aeBlue (right).

ampR

Chromoproteins are convenient reporters

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ampR

Operation Unit 2: Inducible amp resistance

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ampR

Ampicillin resistance is inducible

Growth curve of red construct (+ amp) each in inducer supplemented and inducer free medium. Growth curve of blue construct (+ amp + clavulanic acid) each in inducer supplemented and inducer free medium.

ampR

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ampR

Operation Unit 3: Inducer synthase and transcription factor

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Inducer molecules are produced

ampR

supernatant with inducer + detection strain = bioluminescence supernatant Blue and red construct Detection strains 1 and 2

Winson et al. 1998. FEMS

• Microbiol. Lett. 163:185–192.

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Inducer molecules are produced

ampR

Supernatants containing inducers trigger a bioluminescence signal in detection strains. 50000 100000 150000 200000 250000 300000 350000 400000 realtive light units [-]

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Let‘s try!

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Cross induction is successful

Negative control (inducer free medium) Red construct + supernatant

• f blue construct

Positive control (medium + synthetic inducer) Blue construct + supernatant

• f red construct

Agar diffusion tests

Cross induction of strains

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Negative control (inducer free medium) Red construct + supernatant

• f blue construct

Positive control (medium + synthetic inducer) Blue construct + supernatant

• f red construct

Cross induction is successful

Agar diffusion tests

Cross induction of strains

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Negative control (inducer free medium) Red construct + supernatant

• f blue construct

Positive control (medium + synthetic inducer) Blue construct + supernatant

• f red construct

Cross induction of strains

Cross induction is successful

Agar diffusion tests 21

Cultivation at different ratios

w/o Amp (non-regulated)

[%] 90/10 70/30 50/50 30/70 10/90 Blue construct Red construct

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Cultivation at different ratios

+ Amp (regulated)

Pellets of mixed cultures of blue and red construct after 24 h

• f cultivation with different inoculation ratios.

Blue construct Red construct

w/o Amp (non-regulated)

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[%] 90/10 70/30 50/50 30/70 10/90

Achievements

Entire project out of existing BioBricks Single operation units are functional Created cross induction of constructs Further evaluation of effects on long term cultivation

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There is one more thing …

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Human Practices

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How can we tell people about synthetic biology???

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How to succeed in public relations

An easy guide for all iGEM Teams

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How can we tell people about synthetic biology???

Form a concept – spread the word!

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Create a recognition value!

Strategy

Implemen- tation

Evaluation

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Strategy Evaluation

Form a concept – spread the word!

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Where are our fans from? – Social Media

Strategy Evaluation

Implemen- tation

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When is the best time to post? – Social Media

size = frequency lime green = numerous reactions dark red = little reactions

Mon Tue Wed Thu Fri Sat Sun H

• u

r 4 8 12 16 20 24

Strategy

Evaluatio n Implemen- tation

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Where to place a press release?

Strategy Evaluation

Implemen- tation

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How many people have we reached?

Online 33

Evaluation Strategy

How many people have we reached?

Print Social Media Online 556,598

readers in total

3,243

users / month

6,438

users / month

Strategy Evaluation

Implemen- tation

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How to succeed in public relations

An easy guide for all iGEM Teams

For further information contact iGEM@tu-braunschweig.de 2013.igem.org/Team:Braunschweig www.igem-braunschweig.de

Check it out!

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Instructors: Tobias Unkauf, M.Sc. Jonas Zantow, M.Sc. DSMZ:

• Dr. Christian Jogler

Christian Bödecker, B.Sc. TU Braunschweig:

• Prof. Dr. Stefan Dübel
• Dr. André Frenzel

Acknowledgements

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• E. teamwork -

Engineering a synthetic microbial consortium

• E. teamwork -

Engineering a synthetic microbial consortium

Splitting the work is more efficient

Eiteman, M.A. et al. (2008) A co-fermentation strategy to consume sugar mixtures effectively. J. Biol. Eng. 2, 3.c

Lignocellulose Xylose Glucose + Ethanol + Xylose

Splitting the work is more efficient

Ethanol Lignocellulose Xylose Glucose +

Eiteman, M.A. et al. (2008) A co-fermentation strategy to consume sugar mixtures effectively. J. Biol. Eng. 2, 3.c