TX-TL Workshop, Aug 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:
- Z. Z. Sun, C. A. Hayes, J. Shin, F. Caschera, R. M. Murray, V. Noireaux, 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, An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. ACS Synthetic Biology, 1(1):29–41, 2012.
- Biomolecular Breadboards pages on OpenWetWare
- Modeling overview - setting up simulations using MATLAB-based TX-TL toolbox
- Preliminary data - includes linear DNA protection, protein degradation
- 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)
- ApE (free), Geneious (free trial)
- MATLAB, Simbiology
- 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 detailed schedule for a more complete description of the activities for each day.
Monday amOptional tutorials:
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Tuesday am
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Wednesday am
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Thursday am
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Friday am10:00 am:
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Monday pm
Optional tutorials
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Tuesday pm
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Wednesday pm
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Thursday pm
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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
