TX-TL Workshop, Aug 2013: Detailed Schedule

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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