TX-TL Workshop, Aug 2013: Difference between revisions

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This page contains information for the TX-TL workshop that will take place at Caltech on 26-30 Aug 2013.  This workshop is by invitation only. __NOTOC__
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. __NOTOC__


=== Workshop goals ===
=== Workshop goals ===
By the end of the workshop, participants will be able to:
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)
* Build models and simulate circuits using the TX-TL modeling toolbox (MATLAB-based)
Line 12: Line 12:
=== Travel arrangements ===
=== Travel arrangements ===


Participants are responsible for making their own 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 meeting 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.
* 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.
* [[Directions|Directions and lodging]]
* 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.
* All meetings and lectures will take place in 110 Steele Lab (building 81 on [[directions|campus map]]
* [[Directions|Directions and lodging]] - Saga Motor Hotel is 15 minute walk to campus; Hilton is 15-20 minutes.
* If possible, please bring a laptop with MATLAB and Simbiology installed
* 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).
* If possible, please bring a laptop with Excel (or equivalent) installed.  Other useful software is listed below.


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


The workshop will consist of two sessions each day: morning (9-1) and afternoon (2-6). Participants will be working in the lab, so must have closed shoes and covered legs (no shorts).
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.
* [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 ===
We will make use of the following software as part of the workshop:
* Microsoft Excel (or Numbers)
* [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE] (free), [http://www.geneious.com Geneious] (free trial)
* [http://www.mathworks.com MATLAB], [http://www.mathworks.com/products/simbiology/ Simbiology]
* [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.
 
== Summary Schedule ==
 
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.


{| width=100% border=1
{| width=100% border=1
|- valign=top
|- valign=top
| width=20% |
| width=20% |
==== Monday am ====
Optional tutorials:
* Geneious (Emzo)
| width=20% |
| width=20% |
==== Tuesday am ====
==== Tuesday am ====
* 9:00 am: Analyze TX-TL results from previous day (Clare)
* 9:00 am: [[#TX-TL basics|TX-TL basics]] - Analyze TX-TL results from previous day
* 10:00 am: TX-TL modeling toolbox (Vipul)
* 10:00 am: [[#Extract prep|Extract prep]] - pick colonies
* 10:30 am:  [[#TX-TL basics|TX-TL basics]] - TX-TL modeling toolbox
* 12:00 pm: Lunch
| width=20% |
| width=20% |
==== Wednesday am ====
==== Wednesday am ====
* 9:00 am: Analyze results from previous day
* 10:00 am: Small groups
* 10:00 am: Circuit design and implementation (Shaobin, Clare, Zach, Vincent)
** Ongoing: optional demonstration of pelleting and washing cells (extract prep) in Keck 116
** Students will choose from neg autoregulation, genetic switch, feedfoward loop or logic circuit
* 12:30 pm: Data analysis - Meet in Steele library
** Assembly using Golden Gate; linear + plasmid
* 1 pm: Lunch
| width=20% |
| width=20% |


==== Thursday am ====
==== Thursday am ====
* 9:00 am: Analyze results from previous day and pick colonies
* 10:00 am:  
* 10:00 am: ALL demonstration (1/2 of group)
** G1-G3: [[#Vesicles|Vesicles]], [[#Droplet-based microfluidics|droplets]]
* 10:00 am: Vesicle demonstration (1/2 of group)
** 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 ====
* 9:00 am: Analyze results from previous day
10:00 am:
* 10:00 am: ALL demonstration (1/2 of group)
* [[#Circuit implementation|Circuit implementation]] - analyze and compare data, look at simulations of FFL
* 10:00 am: Vesicle demonstration (1/2 of group)
* 10:30 am: group presentations
* 12:00 pm: Group lunch (Pizza!)
|- valign=top
|- valign=top


| width=20% |
| width=20% |
==== Monday pm ====
==== Monday pm ====
* 1:30 pm, 114 Steele Lab (building 81): Introductions and welcome
* 1:30 pm, 114 Steele Lab (building 81): Introductions and welcome
* 2:00 pm: TX-TL overview + plan for the week (Richard)
* 2:00 pm: TX-TL overview + plan for the week (Vincent, Richard)
* 3:30 pm: Lab session: setting up TX-TL reactions (Clare)
* 3:30 pm: Lab safety session (Clare)
** Lab safety session
* 4:00 pm: [[#TX-TL basics|TX-TL basics]]: setting up TX-TL reactions
** Lab tutorial (for participants who haven't done lab work)
* 5:30 pm: [[#Extract prep|Extract prep]]: streak out colonies
** Set up standard control reactions in TX-TL
* 6:00 pm: done for the day
<hr>
Optional tutorials
* MATLAB/Simbiology (Zoltan)
* Lab techniques (Enoch)
| width=20% |
| width=20% |


==== Tuesday pm ====
==== Tuesday pm ====
* 2:00 pm: Lab session: linear DNA assembly (Zach)
* 1:00 pm: [[#Circuit implementation|Circuit implementation]] - DNA assembly
** Construction of pre-defined circuits using Golden Gate assembly
* 3:00 pm: Breakout discussions (w/ TAs)
* 4:00 pm: TX-TL extract prep overview (Vincent)
* 4:30 pm: [[#Circuit implementation|Circuit implementation]] - run gels; set up overnight runs
* 6:00 pm: done for the day
| width=20% |
| width=20% |


==== 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
 
| width=20% |
| width=20% |
==== Thursday pm ====
==== Thursday pm ====
* 2:00 pm: Lab session: TX-TL extract prep (Vincent)
* 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
| width=20% |
| width=20% |
|}
|}
=== 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
<hr>
* 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 <br>
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
<hr>
* 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
<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, afternoon: get data from plate reader for morning runs; set up new overnight
<hr>
* 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

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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