View source for Mini-bootcamp 2011

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

=== Group ===
<center>
{| width=90%
|- valign=top
| width=25% |
===== Students =====
* Jorge Goncalves
* Jun Liu
* Yuan Ye
* Enoch Yeung
| width=25% |

===== Part-time students =====
* Elisa Franco
* Richard Murray
| width=25% |
===== Instructors =====
* Emzo de los Santos
* Joe Meyerowitz
* Ophelia Venturelli
* Vanessa Jonsson
* Jongmin Kim
| width=25% |

===== Project advisor =====
* Richard Murray
|}
</center>

=== Bootcamp description ===

{| width=100% border=1
|- valign=top
| rowspan=3 |
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
|
[[Image:lambda_switch.png|300px]]
* Circuit layout: directions, ordering
|-
| 
[[Image:biobrick_plasmid.png|300px]]
* Copy number
* Origin of replication
* Antibiotic resistance
* [[http:partsregistry.org/Plasmid_backbones|Biobrick plasmids]]
|-
| 
* (Cell strain)
* Growth media: LB, M9/glycerol, M9/glucose
* Induction level
* Temperature
|}

=== Schedule ===
{| width=100% border=1
|- valign=top
| width=20% | 14 Feb: 2-4
* Biocircuits mini-group meeting - planning
* Richard
| width=20% | 15 Feb
| width=20% | 16 Feb: 
* [[#Session 0: project discussion and lab tour|Session 0: project discussion and lab tour]]
* Richard, Ophelia
* Joe, Elisa
| width=20% | 17 Feb: 2:30-6
* [[Mini-bootcamp_2011/Session 1: lab techniques|Session 1: lab techniques]]
* Emzo, Jongmin
| width=20% | 18 Feb
|- valign=top
| width=20% | 21 Feb: 1-6
* [[#Session 2: cloning|Session 2: cloning]]
* Joe, Emzo
| width=20% | 22 Feb
| width=20% | 23 Feb: 1-6
* [[#Session 3: plate reader|Session 3: plate reader]]
* Ophelia, Richard
| width=20% | 24 Feb
* Project #1
| width=20% | 25 Feb
* Project #1

|- valign=top
| width=20% | 28 Feb
* Project #1
| width=20% | 1 Mar
* Project #1
| width=20% | 2 Mar: TBD
* [[#Session 4: microscopy|Session 4: microscopy]]
* Ophelia, Joe
| width=20% | 3 Mar
*Project #1
| width=20% | 4 Mar
*Project #1
|- valign=top
| width=20% | 7 Mar: TBD
* [[#Session 5: flow cytometry|Session 5: flow cytometry]]
* Joe, Vanessa
| width=20% | 8 Mar: TBD
* [[#Session 6: spectrofluorometer|Session 6: spectrofluorometer]]
* Jongmin, Elisa
| width=20% | 9 Mar
* Project #3
| width=20% | 10 Mar
* Project #3
| width=20% | 11 Mar
* Project #3
|}
&nbsp;
<hr>
==== Session 0: project discussion and lab tour ====
{| width=100%
|- valign=top
| width=50% |
===== Session goals: =====
* Overview of project goals
* Tour of 040 Keck
* Lab safety
===== Instructors: =====
* Richard Murray, Ophelia Venturelli
* Joe Meyerowitz, Elisa Franco (lab tour, safety)
<hr>
==== [[Mini-bootcamp_2011/Session 1: lab techniques|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.
{| width=100%
|- valign=top
| width=50% |
===== Session goals: =====
* Lab safety
* Laboratory techniques: gloves, pipetting, disposal
* Transforming plasmids into cells, growing, extracting
* Clean up: benches, glassware

===== Instructors: =====
* Emzo
* Jongmin 
| width=50% |
===== Techniques and equipment: =====
* Pipetting
* Transformation, selection, growth
* Optical density (OD) measurements (nanodrop?)
* Mini-preps
* Quantification (nanodrop)
* Autoclave
|}
<hr>

==== [[Mini-bootcamp_2011/Session 2: cloning|Session 2: cloning]]  ====
{| width=100%
|- valign=top
| width=50% |
===== 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
| width=50% |

===== Techniques and equipment: =====
* PCR
* Restriction digests
* Gels
* Gibson assembly
|}
<hr>

==== Session 3: [[plate reader]] ====
{| width=100%
|- valign=top
| width=50% |
===== 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
| width=50% |

===== Techniques and equipment: =====
* Victor X3 plate reader
|}
<hr>

==== Session 4: [[microscope]] ====
{| width=100%
|- valign=top
| width=50% |
===== 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
| width=50% |
===== Techniques and equipment: =====
* Olympus IX-81 microscope
* MATLAB tools for image analysis
|}
<hr>

==== Session 5: flow cytometer ====
{| width=100%
|- valign=top
| width=50% |
===== Session goals: =====
* 
===== Instructors: =====
* Joe
* Vanessa
| width=50% |
===== Techniques and equipment: =====
* FACS Calibur flow cytometer
* MaxQuant (?)
|}
<hr>

==== Session 6: [Mini-bootcamp_2011/Session_6:_Spectrofluorometer|spectrofluorometer] ====
{| width=100%
|- valign=top
| width=50% |
===== 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
| width=50% |

===== Techniques and equipment: =====
* PURExpress protein expression kit
* Protein gels (?)
* Fluorilog 3 spectrofluorometer
|}