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Revision as of 19:22, 17 October 2018

SCUT-ChinaA

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 join in this study which aims to standardize the measurements of GFP’s fluorescence. Different from the Fourth International InterLaboratory Measurement Study, this year we measured both OD and colony-forming units (CFUs) to find out whether we can reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or CFUs instead of OD. In this study, we were using plate reader for measuring.

Materials and methods

    1. Plasmid

    All the plasmid located in Kit Plate 7

    1. Negative Control (BBa_R0040): well 2D
    2. Positive Control (BBa_I20270): well 2B
    3. Test Device 1 (BBa_J364000): well 2F
    4. Test Device 2 (BBa_J364001): well 2H
    5. Test Device 3 (BBa_J364002): well 2J
    6. Test Device 4 (BBa_J364007): well 2L
    7. Test Device 5 (BBa_J364008): well 2N
    8. Test Device 6 (BBa_J364009): well 2P

    2. Stains

    E.coil DH5α

    3. Materials

    1. 1ml LUDOX CL-X
    2. 150 μL Silica Bead (microsphere suspension)
    3. Fluorescein (powder, in amber tube)
    4. iGEM Parts Distribution Kit Plates
    5. 1x PBS (phosphate buffered saline, pH 7.4 - 7.6)
    6. ddH2O
    7. LB (Luria Bertani) media
    8. Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
    9. 50 ml Falcon tube (covered in foil to block light)
    10. 1.5 ml eppendorf tubes
    11. Ice bucket with ice
    12. Micropipettes and Micropipette tips
    13. 96 well plates, black with clear flat bottom
    14. 96 well plates, transparent with clear flat bottom

    4. Instruments

    Tecan Infinite M200 Pro

    5. Methods

    1. Calibration protocols
      • Calibration 1: OD600 Reference point - LUDOX Protocol
      • Calibration 2: Particle Standard Curve - Microsphere Protocol
      • Calibration 3: Fluorescence standard curve - Fluorescein Protocol
    2. Cell measurement protocol
    3. Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

Result

    Calibration

    OD600 reference point

    Table 1

    Particle Standard Curve

    Table 2
    Figure 1.1 Particle Standard Curve Figure 1.2 Particle Standard Curve (log scale)

    Fluorescein fluorescence standard curve

    Table 3
    Table 4
    Figure 2.1 Fluorescein Standard Curve Figure 2.2 Fluorescein Standard Curve (log scale)

    Cell measurement

    Table 5
    Table 6
    Table 7

    Colony Forming Units per 0.1 OD600 E. coli cultures

    Table 8

Discussion

In conclusion, the data above demonstrate that we finished our InterLab Study successfully. Through this process, we found that OD varies greatly from one instrument to another, while CFUs depends less on instruments. Besides the instrument difference, different teams may use different optimization when measuring fluorescence (we used the optimization of 40 to get optimal values), which increases the variety of OD measurement. But all teams’ methods of counting colonies and calculating CFUs is generally the same. So we consider that CFUs can be a more normalizing standard to fluorescence measurement instead of OD.