Difference between revisions of "TX-TL Workshop, Aug 2013: Detailed Schedule"

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|-
 
|-
 
| 2:00 || Overview (Richard, Vincent)  
 
| 2:00 || Overview (Richard, Vincent)  
* [http://www.cds.caltech.edu/~murray/talks/breadboards_workshop-26Aug13.pdf Biomolecular breadboards], Richard
+
* [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/breadboards_workshop-26Aug13.pdf Biomolecular breadboards], Richard
* [http://www.cds.caltech.edu/~murray/talks/noireaux_txtl-26Aug13.pdf TX-TL toolbox], Vincent
+
* [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/noireaux_txtl-26Aug13.pdf TX-TL toolbox], Vincent
 
* Schedule for the week, Richard
 
* Schedule for the week, Richard
 
| || ||
 
| || ||
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| 4:00  
 
| 4:00  
 
| Set up reactions for inducible promoter reactions (Zach, Clare)
 
| Set up reactions for inducible promoter reactions (Zach, Clare)
* Use the [http://www.cds.caltech.edu/~murray/talks/TXTL_e18_template.xlsx TX-TL spreadsheet] to set up reactions at 4 different inducer levels
+
* Use the [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/TXTL_e18_template.xlsx TX-TL spreadsheet] to set up reactions at 4 different inducer levels
 
* Pipette reactions into 384 well plate (two columns per team, run in duplicate)
 
* Pipette reactions into 384 well plate (two columns per team, run in duplicate)
 
* Run overnight on plate reader at 29 degC
 
* Run overnight on plate reader at 29 degC
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| 9:00 || Analyze TX-TL results from previous day
 
| 9:00 || Analyze TX-TL results from previous day
 
* Take data off of plate reader, plot in Excel (or MATLAB)
 
* Take data off of plate reader, plot in Excel (or MATLAB)
 
+
* [[Media:firstday_data.xlsx|Data available here]]
 
Golden Gate cloning overview (1 hr, Zach)
 
Golden Gate cloning overview (1 hr, Zach)
 +
* [[Media:overallfirstGenAssembly.png|diagram]]
 
| || ||
 
| || ||
 
|- valign=top
 
|- valign=top
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|- valign=top
 
|- valign=top
 
| 10:30 || TX-TL modeling toolbox (Vipul, Zoltan)
 
| 10:30 || TX-TL modeling toolbox (Vipul, Zoltan)
* Toolbox overview (45 m)
+
* [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/txtl_modeling-27Aug13.pdf Toolbox overview] (45 m)
 
* Hands-on demo (30 m)
 
* Hands-on demo (30 m)
 
* Run models of results from previous evening (script)
 
* Run models of results from previous evening (script)
 +
* [http://sourceforge.net/projects/txtl/?source=dlp Source code] (via SourceForge)
 
| || ||
 
| || ||
 
|-
 
|-
 
| 12:00 || colspan=4 align=center | Lunch
 
| 12:00 || colspan=4 align=center | Lunch
 
|- valign=top
 
|- valign=top
| 1:00 || || DNA assembly (Zach, Clare, Shaobin) [[File:GoldenBraid Assembly 082713.png|20px]]
+
| 1:00 || || DNA assembly (Zach, Clare, Shaobin)  
* Assemble feedforward loop onto individual plasmids ("3X"): AraC, TetR, deGFP
+
* Assemble feedforward loop onto individual plasmids ("3X"): AraC, TetR, deGFP ([[Media:GoldenBraid Assembly 082713.png|diagram]], [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/DNA_Assembly_Protocol.pdf protocol])
 
* Start PCR reactions to amplify linear
 
* Start PCR reactions to amplify linear
 
* Transform plasmids (3X) and streak out on plates
 
* Transform plasmids (3X) and streak out on plates
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* PCR cleanup linear DNA segments
 
* PCR cleanup linear DNA segments
 
* Each group will set up 2 columns of runs (duplicates).  Vary aTc and arabinose (2 each + controls)
 
* Each group will set up 2 columns of runs (duplicates).  Vary aTc and arabinose (2 each + controls)
* Biotek 1: Run linear FFL overnight at 29 degC
+
* Biotek 1: Run linear FFL overnight at 29 degC [[Media:TXTL e11 linear IFFL 082713.xlsx|Spreadsheet available here]]
 
[Pre-prepared linear DNA available for all circuits in case your PCR didn't work]
 
[Pre-prepared linear DNA available for all circuits in case your PCR didn't work]
 
|  
 
|  
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==== Wednesday ====
 
==== Wednesday ====
 
Goals
 
Goals
* TX-TL implement: analyze linear DNA, set up and run 3X plasmids in TX-TL, create 1X plasmids for use on Thursday
+
* TX-TL implement: analyze linear DNA, set up and run 3X plasmids in TX-TL
 
* TX-TL design: create circuits and set up overnight runs
 
* TX-TL design: create circuits and set up overnight runs
 
* TX-TL extract: bead beating and dialysis
 
* TX-TL extract: bead beating and dialysis
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! width=25% align=center | TX-TL design
 
! width=25% align=center | TX-TL design
 
! width=25% align = center | TX-TL extract
 
! width=25% align = center | TX-TL extract
|- valign=top
 
| 9:00 || || Analyze TX-TL results from previous day
 
* Grab and analyze linear DNA results
 
Pick colonies and start cultures for plasmid FFL circuits
 
* Pick colonies for 3X FFL circuits
 
* Set up colony PCR + start cell cultures
 
[Pre-prepared plates available for all circuits in case you didn't get any colonies]
 
| ||
 
 
|- valign=top
 
|- valign=top
 
| 10:00
 
| 10:00
 
| G4, G5: Vesicles prep: phospholipids [Vincent]
 
| G4, G5: Vesicles prep: phospholipids [Vincent]
 
G6: ALL protocol development [Enoch]
 
G6: ALL protocol development [Enoch]
| valign=middle | PCR runs
+
| valign=middle |  
 
| G1-G3: Plan out circuits designs to build and test (with TAs)
 
| G1-G3: Plan out circuits designs to build and test (with TAs)
 
* Decide on what experiments you want to run
 
* Decide on what experiments you want to run
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* Demonstrate how to pellet and wash cells
 
* Demonstrate how to pellet and wash cells
 
|- valign=top
 
|- valign=top
| 12:00 || || Run gels to check colony PCR || ||
+
| 12:30 || || Go over data from previous night [[Media:Workday2 IFFL data 082713.xlsx|Data available here]]  || ||
 
|- valign=top
 
|- valign=top
 
| 1:00 || colspan=4 | Lunch
 
| 1:00 || colspan=4 | Lunch
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| 2:00
 
| 2:00
 
| G4-G6: demonstration sessions (optional)
 
| G4-G6: demonstration sessions (optional)
* Vesicles demo (Vincent): review preparation of feeding, reaction, phospholipids + live vesicles prep [Vincent] + view pre-prepared vesicles on microscope  
+
* 2:00 (prep @ 1:45): Vesicles demo (Vincent): review preparation of feeding, reaction, phospholipids + live vesicles prep [Vincent] + view pre-prepared vesicles on microscope
* Droplets (Enoch): ALL overview + set up reactions for evening run
+
* 3:30: Droplets (Enoch): ALL overview + set up reactions for evening run (Keck 040 - sub-basement)
 +
 
 +
Remaining time: Meet in small groups and work on circuit
 
|  
 
|  
 
|  
 
|  
| G1-G3: bead beating, centrifugation, dialysis (Jongmin, Anu, Clare)
+
| G1-G3: bead beating, centrifugation, dialysis (Jongmin, Anu, Clare) - Keck 116
 
* Each student to set up at least one bead beating tube
 
* Each student to set up at least one bead beating tube
 
* Run bead beating and centrifuge results
 
* Run bead beating and centrifuge results
 
* Load dialysis cassettes
 
* Load dialysis cassettes
|- valign=top
+
 
| 4:00
+
Remaining time: Meet in small groups and work on circuit
|
 
| Prepare plasmid DNA
 
* Mini-prep 3X plasmids
 
* Transform 1X plasmid (from Shaobin) and streak plates
 
[Pre-prepared plasmid and linear versions of all circuits available as a backup]
 
| Circuit design and preparation (if needed)
 
|
 
 
|- valign=top
 
|- valign=top
 
| 5:00
 
| 5:00
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* Start ALL runs (Enoch)
 
* Start ALL runs (Enoch)
 
| colspan=2 | Set up overnight runs
 
| colspan=2 | Set up overnight runs
* Biotek1: FFL 3X plasmids
+
* Biotek1: FFL 3X plasmids [[Media:TXTL e11 linear IFFL workshop test plasmid 082813.xlsx|Spreadsheet available here]]
* Victor: student-designed circuits (linear DNA)
+
* Victor: RNA circuit
 +
* Biotek 2: Switch and logic circuits
 
|
 
|
 
|-  
 
|-  
 
| 6:00 || colspan=4 | Done for the day
 
| 6:00 || colspan=4 | Done for the day
 +
|-
 +
| colspan=5 align=center | Homework: Simulate feedfoward loops + individual circuits
 
|}
 
|}
  
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! width=25% align=center | TX-TL design
 
! width=25% align=center | TX-TL design
 
! width=25% align = center | TX-TL extract
 
! width=25% align = center | TX-TL extract
|- valign=top
 
| 9:00
 
|
 
| Analyze TX-TL results from previous day
 
* Grab and analyze 3X plasmid FFL results
 
Pick colonies and start cultures for plasmid FFL circuits
 
* Pick colonies for 1X plasmid FFL circuit
 
* Set up colony PCR + start cell cultures
 
[Pre-prepared plates of all circuits available as backups]
 
| Analyze TX-TL results from previous day
 
* Grab and analyze linear DNA results
 
|
 
 
|- valign=top
 
|- valign=top
 
| 10:00
 
| 10:00
 
| G1-G3: demonstration sessions (optional)
 
| G1-G3: demonstration sessions (optional)
* Vesicles demo (Vincent): review prep of reaction, feeding, phospholipid, vesicles + view vesicles from previous day on microscope  
+
* 10:00: Vesicles demo (Vincent): review prep of reaction, feeding, phospholipid, vesicles + view vesicles from previous day on microscope  
* Droplets (Enoch): ALL overview + set up reactions for afternoon run
+
* 11:30: Droplets (Enoch): ALL overview + set up reactions for afternoon run (Keck 040 - sub-basement)
 
|  
 
|  
 
| Circuit design and preparation (if needed)
 
| Circuit design and preparation (if needed)
 
* Decide on new runs to be done with circuit variants
 
* Decide on new runs to be done with circuit variants
| G4-G6: bead beating, centrifugation, dialysis (Anu, Jongmin, Clare)
+
| G4-G6: bead beating, centrifugation, dialysis (Anu, Jongmin, Clare) - Keck 116
 
* Each student to set up at least one bead beating tube
 
* Each student to set up at least one bead beating tube
 
* Run bead beating and centrifuge results
 
* Run bead beating and centrifuge results
 
* Load dialysis cassettes
 
* Load dialysis cassettes
 +
|- valign=top
 +
| 12:30 || || Go over data from previous night [[Media:Workday3 IFFL data 082813.xlsx|Data available here]] || ||
 
|- valign=top
 
|- valign=top
 
| 1:00 || colspan=4 | Lunch
 
| 1:00 || colspan=4 | Lunch
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|  
 
|  
 
|
 
|
| Extract prep overview (Clare)
+
| [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/txtl_extract-29Aug13.pdf Extract prep overview] (Clare) - Steele library
* Use slides to go through whole process and calibration procedure
+
* Review of whole process and calibration procedure
 
* Discuss variability between extract batches (with data)
 
* Discuss variability between extract batches (with data)
 
|- valign=top
 
|- valign=top
| 4:00
+
| 3:00 || colspan=4 | Meet in small groups
 +
 
 +
 
 +
 
 +
 
 +
|- valign=top
 +
| 5:00
 
|  
 
|  
| 1X plasmid runs
 
* Mini-prep plasmids; run gel to verify
 
 
In vivo runs
 
In vivo runs
 
* Dilute cells and set up in 96 well plate
 
* Dilute cells and set up in 96 well plate
[Pre-prepared 1X plasmids available as backup]
+
* Run in Biotek 2
 +
|
 
|
 
|
 
|
 
|
 
|- valign=top
 
|- valign=top
| 5:00
+
| 6:00 || colspan=4 | Done for the day
|
 
| colspan=2 | Set up overnight runs
 
* Biotek1: FFL 1X plasmids
 
* Biotek2: FFL in vivo
 
* Victor: student-designed circuits
 
|
 
 
|-
 
|-
| 6:00 || colspan=4 | Done for the day
+
| colspan=5 align=center | Homework: Simulate feedfoward loops + individual circuits
 
|}
 
|}
  
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! width=25% align = center | TX-TL extract
 
! width=25% align = center | TX-TL extract
 
|- valign=top
 
|- valign=top
| 9:00
+
| 10:00
 
|
 
|
 
| Analyze and compare data
 
| Analyze and compare data
 +
* [[Media:Workday4 IFFL in vivo data 082913.xlsx|Data available here]]
 
* Simulations (Tue night), linear TX-TL (Tue night), plasmid TX-TL (Wed/Thu nights), cells (Thu night)  
 
* Simulations (Tue night), linear TX-TL (Tue night), plasmid TX-TL (Wed/Thu nights), cells (Thu night)  
| Analyze data
+
| Analyze data  
* Collect data from plate reader
+
* Data analysis of in vivo FFL results
* Create 10 minute presentation of results (with TAs)
 
 
|
 
|
 
|- valign=top
 
|- valign=top
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| colspan=3 | Group presentations
 
| colspan=3 | Group presentations
 
* 10 minute presentation of circuit results to the group
 
* 10 minute presentation of circuit results to the group
* Enoch: ALL data
+
* Presentations (password protected): [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/private/txtl-group1.pdf G1 (switches)], [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/private/txtl-group2.pdf G2 (logic)], [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/private/txtl-group3.pdf G3 (RNA)], [http://www.cds.caltech.edu/~murray/courses/txtl-aug13/private/txtl-group6.pdf G6 (ALL)]
 
|
 
|
 
|-
 
|-
 
| 12:00
 
| 12:00
| colspan=4 | Group lunch (optional)
+
| colspan=4 | Group lunch - Pizza! Meet in Steele library
 
|}
 
|}

Latest revision as of 19:52, 4 September 2013

The detailed schedule below describes that is happening in each track of the TX-TL workshop and roughly when different groups should be doing different things. Zach and Clare are responsible for keeping track of what is going on. The master copy of this schedule is on the wiki (not publicly accessible). A summary of the workshop schedule is available on the main workshop page.

Monday

Goals:

  • TX-TL basics: Get everyone up to speed on the basics of TX-TL and the tools we will need for the rest of the week
  • TX-TL extract: Create colonies from stocks


Time TX-TL basics TX-TL implement TX-TL design TX-TL extract
9:30 Optional tutorials
  • Geneious (Emzo)
12:00 Lunch at Chandler: meet outside of Keck Lab at noon
1:30 Welcome and Introductions, 114 Steele Lab
2:00 Overview (Richard, Vincent)
3:30 Lab safety session (Clare)
4:00 Set up reactions for inducible promoter reactions (Zach, Clare)
  • Use the TX-TL spreadsheet to set up reactions at 4 different inducer levels
  • Pipette reactions into 384 well plate (two columns per team, run in duplicate)
  • Run overnight on plate reader at 29 degC
5:30 Streak out colonies
6:00 Done for the day
7:30 Optional tutorials
  • MATLAB/Simbiology (Zoltan)
  • Lab techniques (Enoch)

Tuesday

Goals:

  • TX-TL basics: plot experimental data and learn about TX-TL modeling and simulation
  • TX-TL implement: create and run linear DNA, create "3X" plasmid DNA plates
  • TX-TL design: meet with TAs to discuss projects
  • TX-TL extract: pick colonies and grow up cells
  • TX-TL extras: prepare phospholipids for use on Wed


Time TX-TL basics TX-TL implement TX-TL design TX-TL extract
9:00 Analyze TX-TL results from previous day

Golden Gate cloning overview (1 hr, Zach)

10:00 Pick colonies, start cultures
10:30 TX-TL modeling toolbox (Vipul, Zoltan)
12:00 Lunch
1:00 DNA assembly (Zach, Clare, Shaobin)
  • Assemble feedforward loop onto individual plasmids ("3X"): AraC, TetR, deGFP (diagram, protocol)
  • Start PCR reactions to amplify linear
  • Transform plasmids (3X) and streak out on plates
3:00 G4, G5: Vesicles prep. Reaction and feeding solutions [Vincent]

G6: ALL intro session [Enoch]

PCR runs (2 hr) G1-G3: Breakout discussions (w/ TAs)
  • Each group will choose a circuit to prototype using linear DNA on Wed, Thu
  • Choices: RNA circuit, negative auto-regulation, genetic switch, IFFL2
  • TAs: Emzo de los Santos (vesicles), Victoria Hsiao (ALL), Dan Siegal-Gaskins (logic), Vipul Singhal (RNA), Zoltan Tuza (genetic switch), Yong Wu (vesicles)
5:00 Set up linear DNA runs
  • Run gels to confirm PCR results
  • PCR cleanup linear DNA segments
  • Each group will set up 2 columns of runs (duplicates). Vary aTc and arabinose (2 each + controls)
  • Biotek 1: Run linear FFL overnight at 29 degC Spreadsheet available here

[Pre-prepared linear DNA available for all circuits in case your PCR didn't work]

Passage cells
7:00 Done for the day
Homework: Simulate feedfoward loops + individual circuits

Wednesday

Goals

  • TX-TL implement: analyze linear DNA, set up and run 3X plasmids in TX-TL
  • TX-TL design: create circuits and set up overnight runs
  • TX-TL extract: bead beating and dialysis
  • TX-TL extras: set up and run vesicles, set up and run droplets


Time TX-TL extras TX-TL implement TX-TL design TX-TL extract
10:00 G4, G5: Vesicles prep: phospholipids [Vincent]

G6: ALL protocol development [Enoch]

G1-G3: Plan out circuits designs to build and test (with TAs)
  • Decide on what experiments you want to run
  • Check availability of parts; plan out assemblies for the day, if needed
Pellet and wash cells (Jongmin, Anu)
  • Demonstrate how to pellet and wash cells
12:30 Go over data from previous night Data available here
1:00 Lunch
2:00 G4-G6: demonstration sessions (optional)
  • 2:00 (prep @ 1:45): Vesicles demo (Vincent): review preparation of feeding, reaction, phospholipids + live vesicles prep [Vincent] + view pre-prepared vesicles on microscope
  • 3:30: Droplets (Enoch): ALL overview + set up reactions for evening run (Keck 040 - sub-basement)

Remaining time: Meet in small groups and work on circuit

G1-G3: bead beating, centrifugation, dialysis (Jongmin, Anu, Clare) - Keck 116
  • Each student to set up at least one bead beating tube
  • Run bead beating and centrifuge results
  • Load dialysis cassettes

Remaining time: Meet in small groups and work on circuit

5:00
  • Start ALL runs (Enoch)
Set up overnight runs
6:00 Done for the day
Homework: Simulate feedfoward loops + individual circuits

Thursday

Goals

  • TX-TL implement: analyze 3X plasmid DNA circuits, set up and run 1X plasmids in TX-TL + cells with 1X plasmid
  • TX-TL design: modify circuits and/or set up plasmid versions; set up overnight runs
  • TX-TL extract: bead beating and dialysis (second set of groups)
  • TX-TL extras: set up and run vesicles, set up and run droplets (first set of groups)


Time TX-TL extras TX-TL implement TX-TL design TX-TL extract
10:00 G1-G3: demonstration sessions (optional)
  • 10:00: Vesicles demo (Vincent): review prep of reaction, feeding, phospholipid, vesicles + view vesicles from previous day on microscope
  • 11:30: Droplets (Enoch): ALL overview + set up reactions for afternoon run (Keck 040 - sub-basement)
Circuit design and preparation (if needed)
  • Decide on new runs to be done with circuit variants
G4-G6: bead beating, centrifugation, dialysis (Anu, Jongmin, Clare) - Keck 116
  • Each student to set up at least one bead beating tube
  • Run bead beating and centrifuge results
  • Load dialysis cassettes
12:30 Go over data from previous night Data available here
1:00 Lunch
2:00
  • Start ALL runs (Enoch)
Extract prep overview (Clare) - Steele library
  • Review of whole process and calibration procedure
  • Discuss variability between extract batches (with data)
3:00 Meet in small groups



5:00

In vivo runs

  • Dilute cells and set up in 96 well plate
  • Run in Biotek 2
6:00 Done for the day
Homework: Simulate feedfoward loops + individual circuits

Friday

Goals:

  • TX-TL implement: compare simulations, linear, 3X plasmid, 1X plasmid and in vivo results
  • TX-TL design: analyze results and make short presentation
  • TX-TL extras: present data from previous days


Time TX-TL extras TX-TL implement TX-TL design TX-TL extract
10:00 Analyze and compare data
  • Data available here
  • Simulations (Tue night), linear TX-TL (Tue night), plasmid TX-TL (Wed/Thu nights), cells (Thu night)
Analyze data
  • Data analysis of in vivo FFL results
10:30 Group presentations
12:00 Group lunch - Pizza! Meet in Steele library