BE 262 project, 2010

This page contains a description of the BE 262 (bootcamp) project for 2010.

Group

Students: Perrin Considine Ron Lin Dmitry Pushkarov Anu Thubagere TAs: Emzo de los Santos Joe Meyerowitz Ophelia Venturelli Victor X Project advisor: Richard Murray

Baseline project description

The goal of this project is to measure variability in gene expression that is relevant for synthetically designed circuits. The issue that we are trying to understand is now much variability arises for the expression of a given circuit under degrees of freedom that are typically not controlled in synthetic designs: Location and orientation of circuit elements in the plasmid Vectors used for expressing the circuit, including copy number and antibiotic resistance Growth conditions (temperature, oxygen, media, growth phase) To understand how these (and other) factors will affect circuit operation, a simple genetic circuit consisting of 1 or 2 promoters will be built and implemented in a variety of conditions. The dynamic response of the circuit will be measured, including cell-to-cell variability (via flow cytometry or microscopy). Project objectives: Characterize the differences (if any) in mean expression level for a standard circuit (PtetR:GFP) in different backbones, growth media, antibiotic concentrations, inducer levels, etc Characterize differences in expression distributions (if any) using flow cytometry; use cell sorting to check for differences in cell phenotype Construct a simple circuit using different design choices and characterize differences in expression level (and distribution)	Circuit layout: directions, ordering
	Copy number Origin of replication Antibiotic resistance Biobrick plasmids
	(Cell strain) Growth media: LB, M9/glycerol, M9/glucose Induction level Temperature

Schedule

Tuesday: project discussion

Evening: first meeting of project team

• Discuss project and decide on conditions to test
• Place cells in liquid culture and grow up (OV)
  • Will grow all stocks in LB overnight

Wednesday: baseline characterization + circuit design, construction

Rough timeline

• 8:30a: meet in Braun: Joe M to bring over to Keck
• Morning: set up baseline tests on plate reader, perform cloning for new circuits
• Afternoon: continued cloning, baseline circuit analysis on flow cytometer & microscope (if available)

Tasks:

1. Induce baseline cells (Oskar's pTet:GFP) in multiple conditions and test in plate reader (Ophelia)
   • Need to decide on conditions (media, [inducer], [antibiogic])
   • Use Victor X3 plate reader in Keck, with Braun plate reader as a backup
   • Start a run by !0:30a and run for ~6 hours
   • Download data at end of run and plot results in MATLAB (fluorescence versus time and induction level for different contexts)
2. Construction and cloning of synthetic circuit (morning, after plate reader is set up; Joe M)
   • Decide on constructs to be built (using combinations of Pcon:TetR & PtetR:GFP)
   • Work through cloning strategy and get started
   • Need to have circuits ready to transform by evening, for overnight growth on plates
3. Measure selected sets of baseline cells using MoFlo cytometer (afternoon; Emzo)
   • Need to decide whether to run same cells from plate reader or a separate set (or subset)
   • Emzo to contact Frank and arrange a time slot

Notes:

• Activities to take place in Keck, since plate reader is there; Braun as a backup location
• Background (Emzo): work on SLIC-based plasmids (pTet:CFP, pLac:CFP for possible use on Friday)
• Background (Ophelia): work on additional circuits for possible testing on Friday (pTet:GFP, pCon:TetR)
• Background (Richard): work on Calibur (run flow; reproduce Joe's problem with leaking)
• RMM unavailable 1:30-3:30p, 4:30-6p, 7-10p
• Touch base at dinner to decide on what we want to run overnight

Thursday: baseline characterization with varying Y, grow up constructed circuits (overnight)

• Analyze data from Wednesday and decide on any new tests to run
• Induce second round of cells in multiple conditions
• Take new measurements of first and second round cells
  • Microscope time reserved in the afternoon (1-5 pm); need to check if still available
  • Frank available in on Thu afternoon for helping with flow cytometer
• Grow up constructed cells in multiple conditions (overnight)

Friday: final round of characterizations (plus retesting, if needed), presentation preparation

• Send in DNA for sequencing (either before 9:30 am for Fri results or later for Mon results)
• Test newly constructed circuits and/or previous circuits in multiple conditions
• Possible colony PCR if needed to make sure the cloning worked
• Prepare presentation
• TA availability
  • Richard: 9a-10:30a (Keck), 11:30a-12p, 5p-6:30p (Keck)
  • Emzo: 9a-12p, 8p-
  • Ophelia: 9a-2:30p, 4:30p-
  • Joe: 9a-12p, 2p-

Reading

• Stochastic Gene Expression in a Single Cell, Elowitz et al, Science 2002
• Measuring the activity of BioBrick promoters using an in vivo reference standard, Kelly et al, JBE, 2009
• Gene Assembly Papers
  • SLIC
  • Gibson Assembly

Data

Victor run 1, Wed afternoon

• 8 different cell strains:

Col 01: 6A1 (LB) Col 02: 2K3 (LB) Col 03: 4A5 (LB) Col 04: 4K5 (LB) Col 05: 4A5 (LB -> M9u) Col 06: 4K5 (LB -> M9u)

Col 07: 4K5 (M9u) Col 08: 1A2 (LB -> M9u) Col 09: 4A5 (LB -> M9y) Col 10: 4K5 (LB -> M9y) Col 11: 4K5 (M9y) Col 12: 1A2 (LB -> M9u) - repeat from above

• Naming: XYZ (MM -> NN ) = plasmid pSBXYZ with pTetR:GFP, grown up in media MM, transferred and measured in media NN with antibiotic Y (K = kan, A = amp)
  • M9u = Minimal media (M9), with glucose
  • M9y = Minimal media (M9), with 0.4% glycerol

• 12 different aTC concentrations: 150 ng/ml -> 0 ng/ml in 2:1 serial diluation

Row A: 150 ng/ml Row B: 75 ng/ml Row C: 37.5 ng/ml Row D: 18.8 ng/ml

Row E: 9.4 ng/ml Row F: 4.7 ng/ml Row G: 2.4 ng/ml Row H: 0 ng/ml

• Though we lost the non-induced data (baseline) because we aborted without saving, but there seems to be a file with the right timestamps (use with caution)
• 6-8 hours of induced data; cells dried up at end of run; attached file has first 23 runs (out of ~30 total)
• Run 1 files: uninduced (xls), induced (xls; partial run)
• MATLAB: ReadVictor.m - MATLAB script to read and plot plate reader data
• Note: check temperatures in this file; not sure they are 37 degC

Victor run 2, Wed overnight

• Re-run of same backbones/cells from run 1, with aTC starting at 50 ng/ml
• Pre-induction data: 3-5 iterations
• Post-induction data -- 5 interations
• Run stopped; due to system "crash"?
• Run 2 files: uninduced (xls), induced (xls; partial run)

Victor run 3, Thu afternoon

• Induced at 4:45p
• 8 different cell strains:

Col 01: 6A1 (LB) Col 02: 4A5 (LB) Col 03: 4K5 (LB) Col 04: 6A1 (M9u) Col 05: 4A5 (LB -> M9u) Col 06: 4K5 (LB -> M9u)

Col 07: 1A2 (LB -> M9u) Col 08: 1A2 (M9u) Col 09: 4A5 (LB -> M9y) Col 10: 4K5 (LB -> M9y) Col 11: 6A1 (M9y) 1A2 (LB -> M9u) - repeat

• 12 different aTC concentrations: 50 ng/ml -> 0 ng/ml in 2:1 serial diluation
• Run 3 files: uninduced (xls), induced (xls)

Pre-boot camp prep

Preparation

Things that we need to prepare ahead of time:

• Flow cytometer access (done)
• Baseline circuit (Oskar's) in various plasmids and cell strains - Oskar/Ophelia (done)
• SLIC protocol - Emzo/Joe (won't use)
• Decide on circuit components and prepare them - figure out next week (O, J, O, R to meet Fri, latest; done)
• Primers for SLIC based on a some selection of components and multiple locations/orientations (skip)
• Test Oskar's circuits on Victor - week of 30 Aug or 6 Sep (done)

Equipment and supplies

Equipment

• Basic cloning equipment: PCR machine, gel rig, pipettes, etc
• Plate reader: can use Victor X3 from 040 Keck
• Flow cytometer: plan to use RMM FACS Calibur (if working) or HHMI-refurbished MoFlo cytometer
  • Talked to Dave Tirrell on 16 Aug about using this instrument. Need to arrange for training (RMM)
  • Need to figure out what materials (if any) need to be ordered to support likely experiments
  • Might be able to use the new BD FACS Calibur unit if we can get it working (arrived 23 Aug)
  • Could also use the Elowitz lab or Spalding teaching lab Quanta for YFP/GFP (OSV)
• (Optional) Fluorescent microscope for single cell imaging. Need moveable stage + autofocus to be able to look at several pads.

Supplies

• Vectors: ???
  • Can we use current vectors that Oskar is testing? What are they?
  • A (RMM, 24 Aug): Oskar is using 4 plasmids out of the BioBricks library. We should be able to use these
• Cloning: restriction enzymes, ligase, buffers, PCR kits, PCR product kits (QIAquick?), mini-prep kits (QIAGEN?)
  • Need to figure out which restriction enzymes we'll need, especially if they are non-standard
• LB, Amp/Kan plates

Project setup

The following tasks need to be done before bootcamp to make sure that everything is ready for the project:

• Test baseline circuits in plate reader (Oskar will do this as part of SURF)
• Test baseline circuits in flow cytometer (RMM, Emzo, Joe, Ophelia)
• Test baseline circuits in microscope (???)
• Grow up cells with baseline circuits and have available for Wednesday activities

Data for Linda

1. Please provide a project description/abstract no later than Sept. 1st.
   • See description at top of page
2. What equipments do you need? (pipettes, eppendorf tubes, centrifuge, etc.; please note volume/amount as well)
   • Standard equipment for doing cloning (all available in Braun)
   • Will need to grow up cells in different media (LB, minimal) + possibly different temperatures
3. What reagents do you need? (including media; please note volume/amount as well)
   • LB and minimal media for growing up cells - enough for 8-12 runs (?)
   • IPTG and ATc - 500 ul should be enough (?)
   • PCR cleanup kit
   • Mini-prep kit
4. Are there any special items you need (please provide brand, model, part number)? i.e. we would have to place an order for them soon, so they will arrive in time.
   • Access to a flow cytometer (see above)
5. How much lab space do you need?
   • 1 bench should be enough; two would be great
6. Project will starts on Sept. 15th, Wed., do you need space to prepare for your project before Wed.? How much space? What time?
   • We'll need to grow up some baseline cells for testing (based on Oskar H's SURF stocks)
7. What are your microscopy needs? What days? When? How long? Also, whether you need particular microscope capabilities (heated stage, fluorescence, DIC, etc.)
   • If we get the flow cytometer working, we'll probably use that plus a plate reader
   • Might use the microscopes if the students want to explore this path