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
  • Take data off of plate reader, plot in Excel (or MATLAB)

Golden Gate cloning overview (1 hr, Zach)

10:00 Pick colonies, start cultures
10:30 TX-TL modeling toolbox (Vipul, Zoltan)
  • Toolbox overview (45 m)
  • Hands-on demo (30 m)
  • Run models of results from previous evening (script)
12:00 Lunch
1:00 DNA assembly (Zach, Clare, Shaobin) GoldenBraid Assembly 082713.png
  • Assemble feedforward loop onto individual plasmids ("3X"): AraC, TetR, deGFP
  • 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

[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, create 1X plasmids for use on Thursday
  • 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
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]

10:00 G4, G5: Vesicles prep: phospholipids [Vincent]

G6: ALL protocol development [Enoch]

PCR runs 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
12:00 Run gels to check colony PCR
1:00 Lunch
2:00 G4-G6: demonstration sessions (optional)
  • 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
G1-G3: bead beating, centrifugation, dialysis (Jongmin, Anu, Clare)
  • Each student to set up at least one bead beating tube
  • Run bead beating and centrifuge results
  • Load dialysis cassettes
4:00 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)
5:00
  • Start ALL runs (Enoch)
Set up overnight runs
  • Biotek1: FFL 3X plasmids
  • Victor: student-designed circuits (linear DNA)
6:00 Done for the day

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
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
10:00 G1-G3: demonstration sessions (optional)
  • 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
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)
  • Each student to set up at least one bead beating tube
  • Run bead beating and centrifuge results
  • Load dialysis cassettes
1:00 Lunch
2:00
  • Start ALL runs (Enoch)
Extract prep overview (Clare)
  • Use slides to go through whole process and calibration procedure
  • Discuss variability between extract batches (with data)
4:00 1X plasmid runs
  • Mini-prep plasmids; run gel to verify

In vivo runs

  • Dilute cells and set up in 96 well plate

[Pre-prepared 1X plasmids available as backup]

5:00 Set up overnight runs
  • Biotek1: FFL 1X plasmids
  • Biotek2: FFL in vivo
  • Victor: student-designed circuits
6:00 Done for the day

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
9:00 Analyze and compare data
  • Simulations (Tue night), linear TX-TL (Tue night), plasmid TX-TL (Wed/Thu nights), cells (Thu night)
Analyze data
  • Collect data from plate reader
  • Create 10 minute presentation of results (with TAs)
10:30 Group presentations
  • 10 minute presentation of circuit results to the group
  • Enoch: ALL data
12:00 Group lunch (optional)