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).

Reading

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

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 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
  • Outside chance that the new BD FACS Calibur unit will arrive in time to try it out
• (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?
• 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: