TX-TL Workshop, Aug 2013: Difference between revisions

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* A set of optional tutorials will take place on 26 Aug from 9 am to 12 pm for participants who do not have experience in either laboratory techniques, sequence analysis software (aPe, Geneious, etc), or MATLAB programming.  While not required, these tutorials may be helpful for some participants to get more out the workshop.
* A set of optional tutorials will take place on 26 Aug from 9 am to 12 pm for participants who do not have experience in either laboratory techniques, sequence analysis software (aPe, Geneious, etc), or MATLAB programming.  While not required, these tutorials may be helpful for some participants to get more out the workshop.
* [[Directions|Directions and lodging]] - Saga Motor Hotel is 15 minute walk to campus; Hilton is 15-20 minutes.
* [[Directions|Directions and lodging]] - Saga Motor Hotel is 15 minute walk to campus; Hilton is 15-20 minutes.
* All meetings and lectures will take place in 110 Steele Lab (building 81 on [[directions|campus map]]
* All meetings and lectures will take place in 114 Steele Lab (building 81 on {{campus map|campus map}}); lab activities will take place in 136 Keck (building 78)
* Participants will be working in the lab, so must have closed shoes and covered legs (no shorts).
* Participants will be working in the lab, so must have closed shoes and covered legs (no shorts).
* If possible, please bring a laptop with MATLAB and Simbiology installed.
* If possible, please bring a laptop with Excel (or equivalent) installed.  Other useful software is listed below.


=== Read-ahead material ===
=== Read-ahead material ===


We encourage you to take a look at the following material before arriving:
We will assume everyone has watched the following 15 minute video before attending the workshop:
* Z. Z. Sun, C. A. Hayes, J. Shin, F. Caschera, R. M. Murray, V. Noireaux, [http://www.jove.com/video/50762 Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology].  ''Journal. of Visualized Experiments'' (JoVE), e50762, doi:10.3791/50762 (2013).
*: '''Note''': this paper has not yet been released; workshop participants will receive a preprint link via e-mail)
 
If you have time, we also encourage you to take a look at the following material before arriving:
* J. Shin and V. Noireaux, [http://pubs.acs.org/doi/abs/10.1021/sb200016s An ''E. coli'' cell-free expression toolbox: application to synthetic gene circuits and artificial cells].  ''ACS Synthetic Biology'', 1(1):29–41, 2012.
* J. Shin and V. Noireaux, [http://pubs.acs.org/doi/abs/10.1021/sb200016s An ''E. coli'' cell-free expression toolbox: application to synthetic gene circuits and artificial cells].  ''ACS Synthetic Biology'', 1(1):29–41, 2012.
* JoVE video (to be posted)
* [http://openwetware.org/wiki/Biomolecular_Breadboards Biomolecular Breadboards pages on OpenWetWare]
** [http://openwetware.org/wiki/Biomolecular_Breadboards:Models Modeling overview] - setting up simulations using MATLAB-based TX-TL toolbox
** [http://openwetware.org/wiki/Biomolecular_Breadboards:Preliminary_Data Preliminary data] - includes linear DNA protection, protein degradation
** [http://openwetware.org/wiki/Biomolecular_Breadboards:More_Info More info] - additional references and presentations on TX-TL


=== Software ===
=== Software ===
We will make use of the following software as part of the workshop:
We will make use of the following software as part of the workshop:
* Excel
* Microsoft Excel (or Numbers)
* aPe, Geneious
* [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE] (free), [http://www.geneious.com Geneious] (free trial)
* MATLAB, Simbiology
* [http://www.mathworks.com MATLAB], [http://www.mathworks.com/products/simbiology/ Simbiology]
* TX-TL modeling toolbox
* [http://sourceforge.net/projects/txtl/ TX-TL modeling toolbox] (requires MATLAB 2010+ and SimBiology]
Participants are encouraged to install this software on their laptops prior to the workshop, but version of the software will also be available on lab computers.
Participants are encouraged to install this software on their laptops prior to the workshop, but version of the software will also be available on lab computers.


== Schedule ==
== Summary Schedule ==


The workshop will consist of two sessions each day: morning (9-1) and afternoon (2-6).
The schedule below gives an overview of the activities during the workshop.  See the [[TX-TL Workshop, Aug 2013: Detailed Schedule|detailed schedule]] for a more complete description of the activities for each day.


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==== Monday am ====
==== Monday am ====
Optional tutorials:
Optional tutorials:
* MATLAB (Zoltan, Vipul)
* Geneious (Emzo)
* Geneious (Emzo, Joe)
* Lab techniques (Enoch, Zach)
| width=20% |
| width=20% |
==== Tuesday am ====
==== Tuesday am ====
* 9:00 am:  [[#TX-TL basics|TX-TL basics]] - Analyze TX-TL results from previous day
* 9:00 am:  [[#TX-TL basics|TX-TL basics]] - Analyze TX-TL results from previous day
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==== Wednesday am ====
==== Wednesday am ====
* 9:00 am: [[#Circuit implementation|Circuit implementation]] - analyze results, pick colonies, set up PCR
* 10:00 am: Small groups
* 10:00 am: [[#Extract prep|Extract prep]] - pellet and wash cells (demonstration)
** Ongoing: optional demonstration of pelleting and washing cells (extract prep) in Keck 116
* [[#Circuit prototyping|Circuit prototyping]] - construct circuits; set up in plate reader (if time)
* 12:30 pm: Data analysis - Meet in Steele library
* 12 pm: [[#Circuit implementation|Circuit implementation]] - run gels
* 1 pm: Lunch
* 1 pm: Lunch
| width=20% |
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==== Thursday am ====
==== Thursday am ====
* [[#Circuit implementation|Circuit implementation]] - mini-prep plasmids
* 10:00 am:
* [[#Circuit prototyping|Circuit prototyping]] - construct circuits; set up in plate reader
** G1-G3: [[#Vesicles|Vesicles]], [[#Droplet-based microfluidics|droplets]]
** G4-G6: [[#Extract prep|Extract prep]]: bead beating, centrifugation, dialysis
** Remaining time: meet in small groups
* 12:30: Data analysis - meet in Steele library
* 1:00 pm: Lunch
| width=20% |
| width=20% |


==== Friday am ====
==== Friday am ====
* [[#Circuit implementation|Circuit implementation]] - analyze and compare data
10:00 am:
* [[#Circuit implementation|Circuit implementation]] - analyze and compare data, look at simulations of FFL
* 10:30 am: group presentations
* 10:30 am: group presentations
* 12:00 pm: Group lunch (optional)
* 12:00 pm: Group lunch (Pizza!)
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* 5:30 pm: [[#Extract prep|Extract prep]]: streak out colonies
* 5:30 pm: [[#Extract prep|Extract prep]]: streak out colonies
* 6:00 pm: done for the day
* 6:00 pm: done for the day
<hr>
Optional tutorials
* MATLAB/Simbiology (Zoltan)
* Lab techniques (Enoch)
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| width=20% |


==== Tuesday pm ====
==== Tuesday pm ====
* 1:00 pm: [[#Circuit implementation|Circuit implementation]] - DNA assembly (Zach)
* 1:00 pm: [[#Circuit implementation|Circuit implementation]] - DNA assembly
* 3:00 pm: [[#Circuit prototyping|Circuit prototyping]] - Discuss and choose circuits
* 3:00 pm: Breakout discussions (w/ TAs)
* 1:00 pm: [[#Circuit implementation|Circuit implementation]] - DNA assembly (Zach)
* 4:30 pm: [[#Circuit implementation|Circuit implementation]] - run gels; set up overnight runs
* 6:00 pm: done for the day
* 6:00 pm: done for the day
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==== Wednesday pm ====
==== Wednesday pm ====
* 2:00 pm: Transformation of circuits into cells (Clare)
* 2:00 pm:
* 4:00 pm: Lab session: TX-TL extract prep (Vincent)
** G1-G3: [[#Extract prep|Extract prep]]: bead beating, centrifugation, dialysis
** G4-G6: [[#Vesicles|Vesicles]], [[#Droplet-based microfluidics|droplets]]
* 5:00 pm: [[#Circuit implementation|Circuit implementation]] - set up overnight of FFL 3X plasmids - meet in lab
* 6:00 pm: done for the day
 
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==== Thursday pm ====
==== Thursday pm ====
* 2:00 pm: Rotating sessions:
* 2:00 pm: Extract prep overview - meet in Steele library (optional)
** TX-TL extract prep (Anu, Jongmin)
* 3:00 pm: Small groups
** Vesicle-based reactions (Vincent)
* 5:00 pm: In vivo FFL runs - meet in lab
** ALL demonstration (Enoch)
* 6:00 pm: done for the day
* 5 pm: [[#Circuit implementation|Circuit implementation]] - set up runs
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|}
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The workshop will consist of four overlapping topics, run in parallel to each other.
=== Tutorials ===


=== TX-TL basics ===
=== TX-TL basics ===
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Schedule:
Schedule:
* Monday, 9:30 am - noon: Optional tutorials
* Monday, 2-3:30 pm: TX-TL overview
* Monday, 2-3:30 pm: TX-TL overview
* Monday, 3:30-5 pm: set reactions using TX-TL spreadsheet, pipette into 384 well plate, run overnight on plate reader
* Monday, 3:30-5 pm: set reactions using TX-TL spreadsheet, pipette into 384 well plate, run overnight on plate reader
* Monday, 7:30-10 pm: Optional tutorials
<hr>
* Tuesday, 9-10 am: take data off of plate reader, plot in Excel (or MATLAB)
* Tuesday, 9-10 am: take data off of plate reader, plot in Excel (or MATLAB)
* Tuesday, 10:30 am - noon: TX-TL modeling toolbox
* Tuesday, 10:30 am - noon: TX-TL modeling toolbox
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The goal of this topic is to demonstrate how to take circuits from linear DNA to plasmids, and from ''in vitro'' to ''in vivo''.  Participants will learn how to assembly a set of predefined circuits (negative autoregulatory gene, genetic switch, RNA-sensing circuit, feedforward loop) in linear and plasmid forms, and will compare the behavior of the circuit operating in TX-TL (linear, plasmid) with the behavior ''in vivo''.
The goal of this topic is to demonstrate how to take circuits from linear DNA to plasmids, and from ''in vitro'' to ''in vivo''.  Participants will learn how to assembly a set of predefined circuits (negative autoregulatory gene, genetic switch, RNA-sensing circuit, feedforward loop) in linear and plasmid forms, and will compare the behavior of the circuit operating in TX-TL (linear, plasmid) with the behavior ''in vivo''.


Instructors: Zach Sun, Shaobin Guo, Dan Siegal-Gaskins, Zoltan Tuza
Instructors: Vincent Noireaux, Zach Sun, Clare Hayes, Shaobin Guo <br>
TAs: Emzo de los Santos, Victoria Hsiao, Dan Siegal-Gaskins, Vipul Singhal, Zoltan Tuza, Yong Wu


Schedule:
Schedule:
* Tuesday, 1-3 pm: linear and plasmid DNA assembly using Golden Gate cloning and standard parts
* Tuesday, 1-3 pm: linear and plasmid DNA assembly using Golden Gate cloning and standard parts
* Tuesday, 3-5 pm: PCR + transformation
* Tuesday, 3-5 pm: PCR + transformation
** Breakout into groups and meet with TAs to discuss circuit details and plans
* Tuesday, 5-6 pm: run gels and verify products; run overnight on plate reader
* Tuesday, 5-6 pm: run gels and verify products; run overnight on plate reader
** Backup linear DNA available for all circuits in case your PCR didn't work
** Backup linear DNA available for all circuits in case your PCR didn't work
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<hr>
<hr>
* Wednesday, 9-10 am: pick colonies and set up colony PCR, start cultures; analyze data and compare to simulations
* Wednesday, 9-10 am: pick colonies and set up colony PCR, start cultures; analyze data and compare to simulations
* Wednesday, 10 am - 12 pm: breakout discussions
** Discuss results and plan out new experiments to explore variations
* Wednesday, 12-1 pm: run gel on colony PCR to verify results
* Wednesday, 12-1 pm: run gel on colony PCR to verify results
* Wednesday 5-6 pm: set up TX-TL reactions and run overnight on plate reader
<hr>
<hr>
* Thursday, morning: mini-prep part of culture to get plasmids for TX-TL; use rest for ''in vivo'' data; set up runs in plate reader
* Thursday, morning: mini-prep part of culture to get plasmids for TX-TL; use rest for ''in vivo'' data; set up runs in plate reader
* Thursday, afternoon: get data from plate reader
* Thursday, afternoon: get data from plate reader for morning runs; set up new overnight
* Note: timing of activities for Thursday can be adjusted if needed
<hr>
<hr>
* Friday 9-10:30 am: analyze and compare results from linear TX-TL, plasmid TX-TL, cells and simulations
* Friday 9-10:30 am: analyze and compare results from linear TX-TL, plasmid TX-TL, cells and simulations
* Friday, 10:30 am - noon: 15 minute presentation of results to the group
=== Circuit prototyping ===
The goal of this topic is to demonstrate how to use TX-TL modeling and experiments to prototype simple circuits.  Participants will use Golden Gate assembly to construct circuits of their own design.  This topic is a bit more free form and the work should be done during downtime for the other topics.
Instructors: Victoria Hsiao, Emzo de los Santos, Joe Meyerowitz, Yong Wu
Schedule:
* Tuesday, 3-5 pm: break into groups and meet with instructors to pick circuits and decide how to proceed
* Wednesday: design iteration #1.  Create circuits in the morning and run in plate reader during the day.
* Thursday: design iteration #2.  Create circuits in the morning and run in plate reader during the day.
* Friday, 10:30 am - noon: 15 minute presentation of results to the group
* Friday, 10:30 am - noon: 15 minute presentation of results to the group


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* Tuesday morning: pick colonies, start cultures, passage cells
* Tuesday morning: pick colonies, start cultures, passage cells
* Wednesday, 10-12: pellet and wash cells
* Wednesday, 10-12: pellet and wash cells
* Wednesday, 1-5: beat beating demonstrations
* Wed, 2-5 and Thu, 10-1: beat beating demonstrations, dialysis
* Thursday, 10-12: dialyze
* Thursday, 3:30-5 pm: buffer prep
* Thursday, 1-3 pm: buffer prep


=== Extensions ===
=== Extensions ===


==== Vesicles ====
In addition to the main topics listed above, some optional topics will also be available.
 
==== Circuit design ====
 
Goal: perform multiple design cycles on simple circuits using linear DNA:
 
* Genetic switch (Gardner-Collins) - Zoltan
* Two-input logic circuit - Dan
* RNA-based transcription factors (Lucks, Cornell) - Vipul
 
==== TX-TL in vesicles ====
 
Goal: learn how to create phospholipid vesicles containing TX-TL and running experiments with them.


* Instructor: Vincent
* Instructor: Vincent
* TAs: Yong, Emzo


==== Droplet-based microfluidics ====
==== Droplet-based microfluidics ====
Goal: implement experimental protocols for droplet-based microfluidics using a system by Advanced Liquid Logic


* Instructor: Enoch
* Instructor: Enoch
* TA: Victoria

Latest revision as of 01:39, 30 August 2013

As part of the DARPA Living Foundries program, Caltech will be hosting a hands-on workshop on cell-free prototyping and debugging of biological circuits. This page contains information for the TX-TL workshop, which will take place at Caltech on 26-30 Aug 2013. This workshop is by invitation only.

Workshop goals

The purpose of the workshop is to provide participants with a working knowledge of cell-free methods for design of biomolecular circuits, using the TX-TL system developed by Vincent Noireaux at U. Minnesota. By the end of the workshop, participants will be able to:

  • Build models and simulate circuits using the TX-TL modeling toolbox (MATLAB-based)
  • Assemble and test genetic circuits using linear DNA assembly in a single day
  • Implement and test a novel 3-6 promoter circuit in E. coli within a single week
  • Create enough TX-TL extract and buffer for 300+ reactions, shipped to your home institution
  • If time: use artificial cells (vesicles) and droplet-based microfluidics for TX-TL circuits

Travel arrangements

The workshop will begin at 1 pm on 26 Aug (Mon) and will finish at noon on 30 Aug (Fri). Participants are responsible for making their own travel arrangements:

  • The workshop will start at 1:30 pm on 26 Aug (Mon) and end at noon on 30 Aug (Fri). For those who are flying in to LAX, it takes 40-60 minutes to get from LAX to Caltech by taxi. If you are flying into Burbank (BUR), it takes 20-30 minutes.
  • A set of optional tutorials will take place on 26 Aug from 9 am to 12 pm for participants who do not have experience in either laboratory techniques, sequence analysis software (aPe, Geneious, etc), or MATLAB programming. While not required, these tutorials may be helpful for some participants to get more out the workshop.
  • Directions and lodging - Saga Motor Hotel is 15 minute walk to campus; Hilton is 15-20 minutes.
  • All meetings and lectures will take place in 114 Steele Lab (building 81 on campus map); lab activities will take place in 136 Keck (building 78)
  • Participants will be working in the lab, so must have closed shoes and covered legs (no shorts).
  • If possible, please bring a laptop with Excel (or equivalent) installed. Other useful software is listed below.

Read-ahead material

We will assume everyone has watched the following 15 minute video before attending the workshop:

If you have time, we also encourage you to take a look at the following material before arriving:

Software

We will make use of the following software as part of the workshop:

Participants are encouraged to install this software on their laptops prior to the workshop, but version of the software will also be available on lab computers.

Summary Schedule

The schedule below gives an overview of the activities during the workshop. See the detailed schedule for a more complete description of the activities for each day.

Monday am

Optional tutorials:

  • Geneious (Emzo)

Tuesday am

Wednesday am

  • 10:00 am: Small groups
    • Ongoing: optional demonstration of pelleting and washing cells (extract prep) in Keck 116
  • 12:30 pm: Data analysis - Meet in Steele library
  • 1 pm: Lunch

Thursday am

  • 10:00 am:
  • 12:30: Data analysis - meet in Steele library
  • 1:00 pm: Lunch

Friday am

10:00 am:

  • Circuit implementation - analyze and compare data, look at simulations of FFL
  • 10:30 am: group presentations
  • 12:00 pm: Group lunch (Pizza!)

Monday pm

  • 1:30 pm, 114 Steele Lab (building 81): Introductions and welcome
  • 2:00 pm: TX-TL overview + plan for the week (Vincent, Richard)
  • 3:30 pm: Lab safety session (Clare)
  • 4:00 pm: TX-TL basics: setting up TX-TL reactions
  • 5:30 pm: Extract prep: streak out colonies
  • 6:00 pm: done for the day

Optional tutorials

  • MATLAB/Simbiology (Zoltan)
  • Lab techniques (Enoch)

Tuesday pm

Wednesday pm

Thursday pm

  • 2:00 pm: Extract prep overview - meet in Steele library (optional)
  • 3:00 pm: Small groups
  • 5:00 pm: In vivo FFL runs - meet in lab
  • 6:00 pm: done for the day

Tutorials

TX-TL basics

The goal of this topic is to demonstrate how the basic elements of the TX-TL framework are used together. There are three main elements: setting up and running TX-TL reactions, modeling TX-TL reactions, and analyzing TX-TL results.

Instructors: Vincent Noireuax, Clare Hayes, Vipul Singhal

Schedule:

  • Monday, 9:30 am - noon: Optional tutorials
  • Monday, 2-3:30 pm: TX-TL overview
  • Monday, 3:30-5 pm: set reactions using TX-TL spreadsheet, pipette into 384 well plate, run overnight on plate reader
  • Monday, 7:30-10 pm: Optional tutorials
  • Tuesday, 9-10 am: take data off of plate reader, plot in Excel (or MATLAB)
  • Tuesday, 10:30 am - noon: TX-TL modeling toolbox

Circuit implementation

The goal of this topic is to demonstrate how to take circuits from linear DNA to plasmids, and from in vitro to in vivo. Participants will learn how to assembly a set of predefined circuits (negative autoregulatory gene, genetic switch, RNA-sensing circuit, feedforward loop) in linear and plasmid forms, and will compare the behavior of the circuit operating in TX-TL (linear, plasmid) with the behavior in vivo.

Instructors: Vincent Noireaux, Zach Sun, Clare Hayes, Shaobin Guo
TAs: Emzo de los Santos, Victoria Hsiao, Dan Siegal-Gaskins, Vipul Singhal, Zoltan Tuza, Yong Wu

Schedule:

  • Tuesday, 1-3 pm: linear and plasmid DNA assembly using Golden Gate cloning and standard parts
  • Tuesday, 3-5 pm: PCR + transformation
    • Breakout into groups and meet with TAs to discuss circuit details and plans
  • Tuesday, 5-6 pm: run gels and verify products; run overnight on plate reader
    • Backup linear DNA available for all circuits in case your PCR didn't work
  • Tuesday evening (homework): create models for your circuits and run simulations in TX-TL modeling toolbox
  • Wednesday, 9-10 am: pick colonies and set up colony PCR, start cultures; analyze data and compare to simulations
  • Wednesday, 10 am - 12 pm: breakout discussions
    • Discuss results and plan out new experiments to explore variations
  • Wednesday, 12-1 pm: run gel on colony PCR to verify results
  • Wednesday 5-6 pm: set up TX-TL reactions and run overnight on plate reader
  • Thursday, morning: mini-prep part of culture to get plasmids for TX-TL; use rest for in vivo data; set up runs in plate reader
  • Thursday, afternoon: get data from plate reader for morning runs; set up new overnight
  • Friday 9-10:30 am: analyze and compare results from linear TX-TL, plasmid TX-TL, cells and simulations
  • Friday, 10:30 am - noon: 15 minute presentation of results to the group

Extract prep

The main steps of extract preparation will be demonstrated during the week. Because it may not be possible to control our timing precisely, we do not anticipate that we will produce usable extract from this batch, but all steps will be demonstrated in the order that they normally occur.

Instructors: Jongmin Kim, Anu Thubagere

Schedule:

  • Monday afternoon: Streak out colonies
  • Tuesday morning: pick colonies, start cultures, passage cells
  • Wednesday, 10-12: pellet and wash cells
  • Wed, 2-5 and Thu, 10-1: beat beating demonstrations, dialysis
  • Thursday, 3:30-5 pm: buffer prep

Extensions

In addition to the main topics listed above, some optional topics will also be available.

Circuit design

Goal: perform multiple design cycles on simple circuits using linear DNA:

  • Genetic switch (Gardner-Collins) - Zoltan
  • Two-input logic circuit - Dan
  • RNA-based transcription factors (Lucks, Cornell) - Vipul

TX-TL in vesicles

Goal: learn how to create phospholipid vesicles containing TX-TL and running experiments with them.

  • Instructor: Vincent
  • TAs: Yong, Emzo

Droplet-based microfluidics

Goal: implement experimental protocols for droplet-based microfluidics using a system by Advanced Liquid Logic

  • Instructor: Enoch
  • TA: Victoria
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