Team:BFSUICC-China/InterLab


BFSUICC-China

 

 

 

 

 

 

 

 

 

 

 

 

Background

All of the 2018 iGEM teams are invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology. Our team took part in this study which aimed to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. The main task is to make sure if normalizing to absolute cell count or colony-forming units (CFU) can reduce lab-to lab variability in fluorescence measurements. Sally, Celina and Mark have done the experiments in the institute of physics of Peking University using Plate Reader. All members participate in processing data.

Material

 

Host organism

E. coli DH5-alpha strain

Engineered construct



    Steps

  • Calibration

    • Since all instruments are different, we have to make sure we know what the standard value in our own apparatus is by making three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve. The result we got in shown in the following part.



    We use plate reader to measure the following dates.

    Calibration 1: OD600 Reference Point

    experiment data


    final data

    Calibration 2: Particle Standard Curve

    experiment data


    final data


    Calibration 3: Fluorescence Curve



    experiment data
    final data

  • Cell Measurement

    For cell measurement, we transform eight plasmid to agar plat and then did cultivate the culture overnight. Then follow the protocol, we did series dilution and then measure the Abs 600 and fluorescence. After 6h, we measured the Abs600 and fluorescence again


  • Transform plasmids


  • Incubate the remainder of the cultures for 6 hours


  • Take 500µL samples of the cultures at 0 hours and 6 hours.
  • During experiment


  • Result

    At 0 hour

    Fluorescence Raw Readings




    Abs600 Raw Readings
    Final data


    At 6 hour

    Fluorescence Raw Readings

    Final data


    Abs600 Raw Readings

  • Colony Forming Units per 0.1 OD600 E. coli cultures

    It’s a simple way to determine the number of cells in a sample of liquid media Since each colony begins as a single cell, we can determine how many live cells were in the volume of media and obtain a cell concentration for our sample as well.

    dilution 3,4,5


    LB Agar + Cam plates with colonies


    During experiment


  • Result

    OD600 of cell cultures – step1


    Count the colonies

    CFU/mL

 

After the interlab

We were doing this experiment at Peking University with a lot help form a graduate. We also had an opportunity to see a plate reader which is something could rarely be seen a in normal lab. During the 5-day experience, we finally got to know what InterLab is doing. Not surprised, we met some troubles in the lab, but after all the 5-day puzzled, the lab is completed and we found that pretty fulfilling.