Mini-bootcamp 2011

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This page contains a description of the mini-bootcamp run in Feb 2011.

  • NOTE: the main copy of this page and the linked pages have been moved to the biocircuits wiki (password required).

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Cloning - high level timeline

Group

Students
  • Jorge Goncalves
  • Jun Liu
  • Yuan Ye
  • Enoch Yeung
Part-time students
  • Elisa Franco
  • Richard Murray
Instructors
  • Emzo de los Santos
  • Joe Meyerowitz
  • Ophelia Venturelli
  • Vanessa Jonsson
  • Jongmin Kim
Project advisor
  • Richard Murray

Bootcamp description

The goal of this bootcamp 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 2 reporters will be built using different DNA locations and directions, and characterized in a variety of conditions. The dynamic response of the circuit will be measured, including cell-to-cell variability (via flow cytometry and microscopy).

Bootcamp objectives:

  • Project 1: Construct a simple genetic circuit that tests the effects of putting different reporters in different configurations in a plasmid. Characterize the differences (if any) in mean expression level of the circuits, possibly in multiple growth conditions, using a plate reader
  • Project 2: Characterize differences in expression distributions using flow cytometry (FACS Calibur) and fluorescent microscopy
  • Project 3: Perform in vitro testing of the constructs using the PURExpress kit and spectrofluorometer to check for differences in mean expression level

Lambda switch.png

  • Circuit layout: directions, ordering

Biobrick plasmid.png

  • (Cell strain)
  • Growth media: LB, M9/glycerol, M9/glucose
  • Induction level
  • Temperature

Schedule

14 Feb: 2-4
  • Biocircuits mini-group meeting - planning
  • Richard
15 Feb 16 Feb: 17 Feb: 2:30-6 18 Feb
21 Feb: 1-6 22 Feb 23 Feb: 1-6 24 Feb
  • Project #1
25 Feb
  • Project #1
28 Feb
  • Project #1
1 Mar
  • Project #1
2 Mar: TBD 3 Mar
  • Project #1
4 Mar
  • Project #1
7 Mar: TBD 8 Mar: TBD 9 Mar
  • Project #3
10 Mar
  • Project #3
11 Mar
  • Project #3

 


Session 0: project discussion and lab tour

Session goals:
  • Overview of project goals
  • Tour of 040 Keck
  • Lab safety
Instructors:
  • Richard Murray, Ophelia Venturelli
  • Joe Meyerowitz, Elisa Franco (lab tour, safety)

Session 1: lab techniques

This lab session will teach some of the basic techniques that will be used throughout the bootcamp. We assume no background in molecular biology laboratory techniques. By the end of this session, students will be able to transform a plasmid into cells, pick colonies containing the plasmid, grow the cells up to a given optical density, extract the plasmids, and quantify them.

Session goals:
  • Lab safety
  • Laboratory techniques: gloves, pipetting, disposal
  • Transforming plasmids into cells, growing, extracting
  • Clean up: benches, glassware
Instructors:
  • Emzo
  • Jongmin
Techniques and equipment:
  • Pipetting
  • Transformation, selection, growth
  • Optical density (OD) measurements (nanodrop?)
  • Mini-preps
  • Quantification (nanodrop)
  • Autoclave

Session 2: cloning

Session goals:
  • Discussion of PCR, primer design, Gibson assembly, gels
  • Demonstration of PCR
  • Demonstration of Gibson assembly
  • Demonstration of electrophoresis, restriction digestion, mapping
  • Transform Gibson product
Instructors:
  • Joe
  • Emzo
Techniques and equipment:
  • PCR
  • Restriction digests
  • Gels
  • Gibson assembly

Session 3: plate reader

Session goals:
  • Set up dose response with E. coli strains using serial dilution method
  • Learn strategies for measuring dynamics in plate reader
  • Growth in shaker (manual time points)
  • Growth in VictorX3 (automated measurements)
  • Measure gene expression (GFP) and OD (cell mass) using VictorX3 over a period of time
Instructors:
  • Ophelia
  • Richard
Techniques and equipment:
  • Victor X3 plate reader

Session 4: microscope

Session goals:
  • Learn basic principles and design of the Olympus IX-81 light microscope
  • Learn how to make agarose pads for imaging E coli
  • Basics of Micro-Manager software
  • Discuss image analysis
Instructors:
  • Ophelia
  • Joe
Techniques and equipment:
  • Olympus IX-81 microscope
  • MATLAB tools for image analysis

Session 5: flow cytometer

Session goals:
Instructors:
  • Joe
  • Vanessa
Techniques and equipment:
  • FACS Calibur flow cytometer
  • MaxQuant (?)

Session 6: spectrofluorometer

Session goals:
  • Learn basic principles of the Fluorolog 3 spectrofluorometer
  • Learn how to run protein gels
  • Basics of the PURExpress protein expression kit
  • Basics of spectrofluorometer software
Instructors:
  • Jongmin
  • Elisa
Techniques and equipment:
  • PURExpress protein expression kit
  • Protein gels
  • Fluorolog 3 spectrofluorometer