Sortostat

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==Purpose==
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A microfluidic chemostat integrated with a cell sorter, which we call a "sort-o-stat", will enable more complicated selections to be applied to a population of cells in continuous culture.  In particular, time varying selective pressures as well as very specific selection strengths can be applied. We will evaluate whether or not these more sophisticated selective pressures will be of practical use in service of evolving engineered biological systems.  Selection can be based on any characteristic that can be reliably measured via microscopy. Lastly, since this is a physical selection (rather than a chemical one), it may be more difficult for cells to find unexpected methods to evade selection.
==Design==
==Design==
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[[Image:SortoStatDesign.PNG|thumb|500px|left|Chip design with sorting chamber displayed in lower right]]
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<br style="clear:both" />
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[[Sortostat/Optimal sorting cutoffs|Optimal sorting cutoffs]]
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[[Sortostat/Design/v2.0]]
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The design of the Sortostat is an extension of the microchemostat design created by Frederick Balagadde. ([http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15994559&query_hl=1&itool=pubmed_docsum REF])
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A proposal for a somewhat similar device is:
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Gordon, R. 1996. Computer controlled evolution of diatoms: design for a compustat. Nova Hedwigia 112: 213-216.
==Protocols==
==Protocols==
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[[Operate Microfluidic Chemostat]]
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[[SortoStat/Setting up a new chip|Setting up a new chip]]
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[[SortoStat/Operation]]
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[[Microfluidic chemostat labview program]]
[[Microfluidic chemostat labview program]]
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[[Sortostat/Image processing|Image Processing]]
==Known Issues==
==Known Issues==
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This section describes common failure points to help in debugging.
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#Apply too high of a dilution rate too quickly
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#*usually a good idea to grow the cells with a very low dilution rate (e.g. D=0.05) for ~10hrs in order to ensure that the cells are growing well and that there are no problems with the chip.  If the cells grow well then you can increase the dilution rate.
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#Cells escaping from the sorting chamber
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#*This can happen as a result of closing both the ClockWise and CounterClockWise valves, resulting in a pressure build up in the chambers between push-UP valves.  It is not clear how pervasive a problem this is, but I have made changes to the [[Microfluidic chemostat labview program]] to close the CW and CCW sequentially to help the problem.
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#Cells stop producing fluorescent proteins
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#Control lines
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#*Over time the control lines empty of water.  This was observed with green food dye.
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#*This problem can be addressed with teflon tubing says the foundry.
==Experimental Results==
==Experimental Results==
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[[SortoStat experiments]]
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*[[Sortostat/Experiments/Early experiments]]
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*[[Sortostat/Experiments/Brian growth tests]]
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*[[Sortostat/Experiments/Two reactor chip]]
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*[[Sortostat/Experiments/Comparison of CFP and YFP cells growth rate]]
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==Posters==
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[[:Image:JK ICSB 2005.pdf|ICSB 2005 Poster]]
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==Notebook==
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*[[Sortostat/Notebook]] - this is where ongoing work with the sortostat is stored.
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==Researchers==
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[[Jason Kelly]], [[Josh Michener]], [[Bryan Hernandez]]

Current revision

Contents

Purpose

A microfluidic chemostat integrated with a cell sorter, which we call a "sort-o-stat", will enable more complicated selections to be applied to a population of cells in continuous culture. In particular, time varying selective pressures as well as very specific selection strengths can be applied. We will evaluate whether or not these more sophisticated selective pressures will be of practical use in service of evolving engineered biological systems. Selection can be based on any characteristic that can be reliably measured via microscopy. Lastly, since this is a physical selection (rather than a chemical one), it may be more difficult for cells to find unexpected methods to evade selection.

Design

Chip design with sorting chamber displayed in lower right
Chip design with sorting chamber displayed in lower right


Optimal sorting cutoffs

Sortostat/Design/v2.0

The design of the Sortostat is an extension of the microchemostat design created by Frederick Balagadde. (REF)

A proposal for a somewhat similar device is:

Gordon, R. 1996. Computer controlled evolution of diatoms: design for a compustat. Nova Hedwigia 112: 213-216.

Protocols

Setting up a new chip

SortoStat/Operation

Microfluidic chemostat labview program

Image Processing

Known Issues

This section describes common failure points to help in debugging.

  1. Apply too high of a dilution rate too quickly
    • usually a good idea to grow the cells with a very low dilution rate (e.g. D=0.05) for ~10hrs in order to ensure that the cells are growing well and that there are no problems with the chip. If the cells grow well then you can increase the dilution rate.
  2. Cells escaping from the sorting chamber
    • This can happen as a result of closing both the ClockWise and CounterClockWise valves, resulting in a pressure build up in the chambers between push-UP valves. It is not clear how pervasive a problem this is, but I have made changes to the Microfluidic chemostat labview program to close the CW and CCW sequentially to help the problem.
  3. Cells stop producing fluorescent proteins
  4. Control lines
    • Over time the control lines empty of water. This was observed with green food dye.
    • This problem can be addressed with teflon tubing says the foundry.

Experimental Results

Posters

ICSB 2005 Poster

Notebook

Researchers

Jason Kelly, Josh Michener, Bryan Hernandez

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