Team:GreatBay China/Interlab

GBC-Interlab


mCATNIP2018@outlook.com
@Igem2018C

Introduction of Interlab

Reliable and repeatable measurement plays an important role as an engineering discipline in synthetic biology. The InterLab Study aims to identify and correct the sources of systematic variability in synthetic biology measurement, thus to improve the measurement tools and methods which can enable labs to reliably build upon others’ work. It will promote the collaboration within the synthetic biology community. Hence, it may propel the synthetic biology to achieve its full potential.

The previous InterLab studies showed that measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule can greatly reduce the variability in different labs’ measurements.

The Fifth Year InterLab’s goal is to remove another source of variability, the number of cells in the sample, by using new cell-counting method.


GreatBay_China investigation had helped in answering the question:Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?




Supplies and Reagents


DNA/Plasmids


  1. Negative Control: BBa_R0040
  2. Positive Control: BBa_I20270
  3. Test Device 1: BBa_J364000
  4. Test Device 2: BBa_J364001
  5. Test Device 3: BBa_J364002
  6. Test Device 4: BBa_J364007
  7. Test Device 5: BBa_J364008
  8. Test Device 6: BBa_J364009

Apparatus


  • 96 well plates, black with clear flat bottom (provided by Bluepha Lab)
  • Plate reader
  • Flow Cytometry
  • Incubator at 37˚C
  • Ice bucket with ice
  • Foil covered 50 ml tube
  • Eppendorf tubes
  • Micropipettes
  • Micropipette tips

Materials


  • Measurement Kit
    • - LUDOX CL-X
    - Silica beads
    - Fluorescein
  • 1x PBS (Phosphate buffered saline, pH 7.4-7.6)
  • ddH2O
  • Competent cells (Escherichia coli strain DH5α)
  • LB (Luria Bertani) media
  • Chloramphenicol
  • LB plates
  • distilled water



Protocols


We had been asked to follow the protocols provided by iGEM HQ.

2018 InterLab Plate Reader Protocol
2018 InterLab Flow Cytometry Protocol
Help: Protocols/Transformation



Members


Charles Wei

Sean Yao

David Yang



Field Work


For the reason that our plate reader could only measure the absorbance, we collaborated with SZU-China and borrowed their plate reader.


Day 1


  • The plasmids from distribution kit had been resuspended and transformed into Escherichia coli DH5α

Day 2


  • Single colonies had been picked from transformation plates and inoculated into LB medium
  • All the supplies and reagents for Calibration and Cell measurement had been prepared

Day 3


  • The three calibration had been done during the dilution of overnight cultures
  • The Cell measurement had been done following the protocol
  • The flow cytometer collected the samples after measured each plate
  • Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures had been done

Day 4


  • Count the colonies (thanks the ColonyCount from promega)




Results


Calibrations


OD600 reference point:



Particle Standard Curve:



Fluorescein Standard Curve:




Raw Plate Reader Measurements


Fluorescence Raw



Abs600 Raw








Our data was accepted on 30th July. Congratulations!


Acknowledgement