Difference between revisions of "Team:MIT/InterlabStudy"

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'''2015 MIT IGEM TEAM'''
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{{Team:MIT/Header}}
  
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'''Introduction'''
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The iGEM Interlab Study aims “is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world”. It is an opportunity for teams throughout the world to build and characterize parts. The purpose of the study as a whole is to be able to “test the consistency of the teams’ data of the measured devices”.
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</style>
  
  
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<body>
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<div>
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<img class = "headerimage" src = "https://static.igem.org/mediawiki/2015/a/ab/Team-MIT-Psbanner.jpg">
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</div>
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<div id = "spacer">
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<div id = "title">
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Interlab Study
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</div>
  
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<div class = "subtitle">
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Introduction
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</div>
  
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<div class = "text" align = "center">
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The iGEM Interlab Study aims “is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world”. It is an opportunity for teams throughout the world to build and characterize parts. The purpose of the study as a whole is to be able to “test the consistency of the teams’ data of the measured devices”.
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</div>
  
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<div class = "subtitle">
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Devices Built and Measured: (Chassis : E coli)
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</div>
  
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'''Devices Built and Measured: (Chassis : E coli)'''
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<div class = "text" align = "center">
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Device 1 : J23101 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates)
  
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Device 1 : J23101 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates)
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Device 2 : J23106 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates)
  
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Device 2 : J23106 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates)
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Device 3 : J23117 + I13504 (B0034-E0040-B0015) -> backbone: PSB1C3 (3 biological replicates)
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</div>
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<div class = "subtitle">
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Measurement Controls:
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</div>
  
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Device 3 : J23117 + I13504 (B0034-E0040-B0015) -> backbone: PSB1C3 (3 biological replicates)
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<div class = "text" align = "center">
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Positive Control: I20270 PSB1C3 -> J23151 + I20270(B0032-E0040-B0010-B0012)
  
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Negative Control: BBa_R0040
  
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Negative Control : NEB 10 B Competent Cells
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</div>
  
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<div class = "subtitle">
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Protocols:
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</div>
  
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'''Measurement Controls:'''
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<div class = "text" align = "center">
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DNA Kit Plate Instructions  (http://parts.igem.org/Help:2015_DNA_Distribution)
  
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Positive Control: I20270 PSB1C3 -> J23151 + I20270(B0032-E0040-B0010-B0012)
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Transformation (http://parts.igem.org/Help:Protocols/Transformation)
  
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Negative Control: BBa_R0040
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Making Liquid cultures:
  
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Negative Control : NEB 10 B Competent Cells
 
  
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• Prepare culture in a 15 mL, round bottom tube.
  
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• Add 5mL LB using a seriological pipette
  
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'''Protocols:'''
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• Add 5uL of 1000x antibiotic (Chloramphenicol.)
  
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DNA Kit Plate Instructions  (http://parts.igem.org/Help:2015_DNA_Distribution)
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• Pick colony using a 10 ul pipette tip on a p2. Eject tip into tube (tip should remain in tube).
  
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Transformation (http://parts.igem.org/Help:Protocols/Transformation)
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• if growing from another liquid culture, 100 uL should be plenty (replacing the 1 colony). Almost no amount is too small, just ensure that you get cells.
  
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Making Liquid cultures:
 
  
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<a href = "http://www.hawaii.edu/microbiology/MO/docs/diversity/Qia-Miniprep.pdf">Miniprep – using Qiagen protocol</a>
  
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• Prepare culture in a 15 mL, round bottom tube.
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<a href = "http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">3 Antibiotic Assembly protocol</a>
  
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• Add 5mL LB using a seriological pipette
 
  
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• Add 5uL of 1000x antibiotic (Chloramphenicol.)
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<a href = "http://parts.igem.org/Help:Protocols/Transformation">Transformation</a>
  
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• Pick colony using a 10 ul pipette tip on a p2. Eject tip into tube (tip should remain in tube).
 
  
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• if growing from another liquid culture, 100 uL should be plenty (replacing the 1 colony). Almost no amount is too small, just ensure that you get cells.
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<a href = "https://www.neb.com/~/media/NebUs/Files/Application%20Notes/appNoteM0486.pdf">Colony PCR (selecting transformants from the transformation plates)</a>
  
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Making Liquid Culture (see above)
  
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Miniprep – using Qiagen protocol (http://www.hawaii.edu/microbiology/MO/docs/diversity/Qia-Miniprep.pdf)
 
  
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3 Antibiotic Assembly protocol – (http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf))
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Generalized E coli Flow Cytometry Protocol( created by Nicholas Delateur Weiss Lab MIT)
  
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1. Grow an overnight in rich media with appropriate antibiotic from a single colony (in previous step)
  
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Transformation (http://parts.igem.org/Help:Protocols/Transformation)
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2. Subculture 125 uL of saturated culture into 5 mL of M9 Minimal Media (below) supplemented with Glycerol, add appropriate antibiotic
  
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3. Grow with shaking at desired temperature until desired OD (0.3 for exponential phase, 1.0ish for steady phase)
  
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Colony PCR (selecting transformants from the transformation plates) – (https://www.neb.com/~/media/NebUs/Files/Application%20Notes/appNoteM0486.pdf)
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4. Dilute 10 uL into 990 uL 1X PBS
  
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5. Interrogate by flow cytometry
  
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Making Liquid Culture (see above)
 
  
  
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Generalized E coli Flow Cytometry Protocol( created by Nicholas Delateur Weiss Lab MIT)
 
  
−
1. Grow an overnight in rich media with appropriate antibiotic from a single colony (in previous step)
 
  
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2. Subculture 125 uL of saturated culture into 5 mL of M9 Minimal Media (below) supplemented with Glycerol, add appropriate antibiotic
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M9 Min w/ Glycerol
  
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3. Grow with shaking at desired temperature until desired OD (0.3 for exponential phase, 1.0ish for steady phase)
 
  
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4. Dilute 10 uL into 990 uL 1X PBS
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• 20 mL 5X m9
  
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5. Interrogate by flow cytometry
 
  
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• 3.4 mL 10mg/mL thiamine
  
  
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• 0.8 mL 50% glycerol
  
  
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M9 Min w/ Glycerol
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• 2 mL 10% cas AA
  
  
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• 20 mL 5X m9
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• 0.2 mL  1 M MgSO4
  
  
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• 3.4 mL 10mg/mL thiamine
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• 10 uL 1 M CaCl2
  
  
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• 0.8 mL 50% glycerol
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Mix, and then add DI water until the total volume is 100 mL (for a total of 100 mL)
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</div>
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<div class = "subtitle">
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Measuring in flow cytometer
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</div>
  
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<div class = "text" align = "center">
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1)    measured beads
  
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• 2 mL 10% cas AA
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2) measured 3 types of devices
  
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3)    measured positive control : I20270 (GFP construct)
  
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• 0.2 mL  1 M MgSO4
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4)      measure negative controls : Untransformed competent cell and  R0040
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</div>
  
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<div class = "subtitle">
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Sequencing
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</div>
  
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• 10 uL 1 M CaCl2
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<div class = "text" align = "center">
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<a href = "https://static.igem.org/mediawiki/2015/2/29/Sequencing_Interlab_Data_final.pdf">
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Final Data
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</a>
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</div>
  
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</div>
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</div>
  
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Mix, and then add DI water until the total volume is 100 mL (for a total of 100 mL)
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</body>
  
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</html>
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'''Measuring in flow cytometer'''
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1)    measured beads
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2) measured 3 types of devices
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+
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3)    measured positive control : I20270 (GFP construct)
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−
 
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4)      measure negative controls : Untransformed competent cell and  R0040
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'''Sequencing'''
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[[File:Sequencing Interlab Data final.pdf]]
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Revision as of 15:23, 3 September 2015


Interlab Study
Introduction
The iGEM Interlab Study aims “is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world”. It is an opportunity for teams throughout the world to build and characterize parts. The purpose of the study as a whole is to be able to “test the consistency of the teams’ data of the measured devices”.
Devices Built and Measured: (Chassis : E coli)
Device 1 : J23101 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates) Device 2 : J23106 + I13504 (B0034-E0040-B0015) -> backbone:PSB1C3 (3 biological replicates) Device 3 : J23117 + I13504 (B0034-E0040-B0015) -> backbone: PSB1C3 (3 biological replicates)
Measurement Controls:
Positive Control: I20270 PSB1C3 -> J23151 + I20270(B0032-E0040-B0010-B0012) Negative Control: BBa_R0040 Negative Control : NEB 10 B Competent Cells
Protocols:
DNA Kit Plate Instructions (http://parts.igem.org/Help:2015_DNA_Distribution) Transformation (http://parts.igem.org/Help:Protocols/Transformation) Making Liquid cultures: • Prepare culture in a 15 mL, round bottom tube. • Add 5mL LB using a seriological pipette • Add 5uL of 1000x antibiotic (Chloramphenicol.) • Pick colony using a 10 ul pipette tip on a p2. Eject tip into tube (tip should remain in tube). • if growing from another liquid culture, 100 uL should be plenty (replacing the 1 colony). Almost no amount is too small, just ensure that you get cells. Miniprep – using Qiagen protocol 3 Antibiotic Assembly protocol Transformation Colony PCR (selecting transformants from the transformation plates) Making Liquid Culture (see above) Generalized E coli Flow Cytometry Protocol( created by Nicholas Delateur Weiss Lab MIT) 1. Grow an overnight in rich media with appropriate antibiotic from a single colony (in previous step) 2. Subculture 125 uL of saturated culture into 5 mL of M9 Minimal Media (below) supplemented with Glycerol, add appropriate antibiotic 3. Grow with shaking at desired temperature until desired OD (0.3 for exponential phase, 1.0ish for steady phase) 4. Dilute 10 uL into 990 uL 1X PBS 5. Interrogate by flow cytometry M9 Min w/ Glycerol • 20 mL 5X m9 • 3.4 mL 10mg/mL thiamine • 0.8 mL 50% glycerol • 2 mL 10% cas AA • 0.2 mL  1 M MgSO4 • 10 uL 1 M CaCl2 Mix, and then add DI water until the total volume is 100 mL (for a total of 100 mL)
Measuring in flow cytometer
1) measured beads 2) measured 3 types of devices 3) measured positive control : I20270 (GFP construct) 4) measure negative controls : Untransformed competent cell and R0040
Sequencing
Final Data