Mini-bootcamp 2011

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).

Quick Links

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	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

14 Feb: 2-4 Biocircuits mini-group meeting - planning Richard	15 Feb	16 Feb: Session 0: project discussion and lab tour Richard, Ophelia Joe, Elisa	17 Feb: 2:30-6 Session 1: lab techniques Emzo, Jongmin	18 Feb
21 Feb: 1-6 Session 2: cloning Joe, Emzo	22 Feb	23 Feb: 1-6 Session 3: plate reader Ophelia, Richard	24 Feb Project #1	25 Feb Project #1
28 Feb Project #1	1 Mar Project #1	2 Mar: TBD Session 4: microscopy Ophelia, Joe	3 Mar Project #1	4 Mar Project #1
7 Mar: TBD Session 5: flow cytometry Joe, Vanessa	8 Mar: TBD Session 6: spectrofluorometer Jongmin, Elisa	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