Want a more interesting circuit? Elowitz et al ., Nature (2000). ? - - PowerPoint PPT Presentation

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Want a more interesting circuit? Elowitz et al ., Nature (2000). ? - - PowerPoint PPT Presentation

Oct 27, 2023 387 likes •773 views

Want a more interesting circuit? Elowitz et al ., Nature (2000). ? lacI tetR cI ? ? Introducing Our Transcription Factor Development Pipeline: Select mutants Using Lysis The idea: release DNA of valid mutants. Implementation: BL21

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Want a more interesting circuit?

Elowitz et al., Nature (2000).

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lacI cI

tetR

? ? ?

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Introducing… Our Transcription Factor

Development Pipeline:

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Select mutants Using Lysis

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The idea: release DNA of valid mutants.

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Lysis cassette from Berkeley 2008: BBa_K112808

BL21 cells have IPTG-inducible T7 RNA Polymerase

Implementation:

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To prove it can work, we need to:

1) Control lysis 2) Recover DNA 3) Select the right DNA

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Control Lysis

Recover DNA Select the right DNA IPTG addition 0 µM IPTG 1000 µM IPTG

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Lysis Control Lysis Control

Control

Lysis

Transformation qPCR Control Lysis

Recover DNA

Select the right DNA

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Transformation qPCR

RFP GFP RFP GFP Lysing (RFP) and control (GFP) cells

Control Lysis Recover DNA

Select the right DNA

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Select mutants Using Lysis

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Characterise in vitro using

microfluidics

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WT

Tetracyclin Repressor

TetO sequence:

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E37A W43S T141A V36F P39K WT

We mutate the sites

  • f DNA interaction

Ramos et al. (2005), Orth et al. (2000), Helbl et al

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In vitro TF characterization

  • 768 TF-DNA interaction

measurements in 5h

  • High sensitivity
  • Absolute affinities

Maerkl et al., 2007 38000 datapoints collected this summer

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enoLOGO: Workman et al, 2005

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Characterise in vitro using

microfluidics

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Characterise in vivo using

reporter genes

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In vivo readout principle

…detect and quantify TetR binding to its promoter

Strong

interaction?

Weak

interaction?

No

Interaction?

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TetR represses RFP: Adding ATC cancels repression:

Readout implementation

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Maximum ATC: 25’000 RFUs No ATC: 2500 RFUs

Wild-type characterisation

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The mutants repress RFP differently:

E37A W43S T141A

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Consistency of the two characterisations:

TetR Mutant In vitro specificity In vivo activity Strong activity Same as WT Same as WT No activity

E37A W43S T141A

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Characterise in vivo using

reporter genes

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Using the

right tools

helps.

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Alberta 2009 Biotec Dresden 2009 MIT 2010 Strasbourg 2010

Microfluidic systems are gaining popularity within iGEM: Maybe your successors will give it a go?

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Thankfully, you can

  • rder from a foundry,

such as Stanford’s.

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In summary, we:

 Set up a lysis-based selection system Developed characterisation methods

  • MITOMI chip for binding specificities
  • RFP-based in vivo system

Demonstrated them with TetR mutants Promoted microfluidics to the iGEM community

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Huge thanks to:

The artist: Our teaching assistants:

Alina Henrike Irina Matt

Our professors:

Bart Deplancke Sebastian Maerkl

Laboratory for Systems Biology and Genetics Laboratory for Biological Network Characterization

Fundraising:

Solange Richter Sacha Sidjanski &Caroline O’Neill

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Questions?

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