Difference between revisions of "Team:MIT/InterlabStudy"

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”.

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)

Positive Control: I20270 PSB1C3 -> J23151 + I20270(B0032-E0040-B0010-B0012)
Negative Control: BBa_R0040
Negative Control : Lucigen 10G Competent Cells

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)

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

Final Data

Flow Cytometer Data

Sample Graphs

J23101+ GFP	J23106 + GFP	J23117 + GFP

Our study proved 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 (in the middle), 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 very close to the output of the same promoter in the Anderson Promoter Study. Given that these are arbitrary units(usually inconsistent), we also noticed huge differences in the output for the J23101 and J23106 promoters, between our study and the Anderson Promoter Study. Despite having the expected order of strength (relative strength) as described above, these huge differences in data probably account for the ambiguous nature of arbitrary units of fluorescence.