BE 262 project, 2010

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

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).

Rough timeline:

• Monday: discuss project with boot camp participants
• Tuesday evening: first meeting of project team
  • Discuss project and decide on circuit + conditions to test
  • Place cells in liquid culture and grow up

• Wednesday: baseline characterization w/ varying X, circuit construction
  • Induce baseline cells (Oskar's pTet:GFP) in multiple conditions and test in plate reader, flow cytometer
    • Use Victor X3 plate reader in Keck, with Braun plate reader as a backup
    • Use MoFlo cytometer (Frank available am and pm; pm likely), with Calibur possible if working. Quanta's as backup
  • Construction and cloning of synthetic circuit (morning)
    • Try SLIC and/or Gibson (based on Emzo experiments)
    • Have available a couple of DNA sequences that can be connected in different ways (pTet:GFP, pCon:TetR)
  • Design primers for sequencing and submit to IDT

• 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 in the afternoon?
  • Grow up constructed cells in multiple conditions (overnight)
    • Frank available in on Thu afternoon for helping with flow cytometer

• 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

Reading

• Intrinsic and extrinsic noise paper (Elowitz)
• Standard promoter paper (Endy lab)

Preparation

Things that we need to prepare ahead of time:

• Primers for SLIC based on a some selection of components and multiple locations/orientations

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