Team:MIT/InterlabStudy


Introduction
The iGEM Interlab Study's aim 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)
E coli Strain : Lucigen 10G
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 : Lucigen 10G 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)




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 (Phosphate Buffered Saline) Solution
  5. Interrogate by flow cytometry

M9 Min w/ Glycerol Recipe

  • 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(Spherotech RCP(Rainbow Calibration Particles)-30-5A). (These are used "for the routine calibration of the channels in flow cytometers"(Spherotech)).
  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
Results

Flow Cytometer Data

Sample Graphs

J23101+ GFP J23106 + GFP J23117 + GFP
Conclusion
Our study showed that among the 3 promoters (J23101, J23106, J23117), the J23101 Promoter was the strongest as it had the highest GFP fluorescence associated with it. In order of strength, the J23101 Promoter was the highest, the J23106 Promoter was moderate, and the J23117 Promoter was the lowest. This is in alignment with the 2006 UC Berkeley "Anderson Promoter" Study (which measured RFP fluorescence). Also, we noticed that the J23117 promoter's output was close to that of the same promoter in the Anderson Promoter Study. It was striking that the output was close for the two given that both of the studies were performed under different conditions. We also noticed that our study's fluorescence difference between J23101 and J23106 was about 3X greater than that of the Anderson Study and the fluorescence difference between J23106 and J23117 was about 6X greater than that of the Anderson Study.