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Revision as of 17:36, 16 October 2018

Section Sample

InterLab

I. Background

“All of 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 is aimed to figure out and correct the sources of systematic variability in synthetic biology measurements. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.

II. Design

Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be a real-time monitor without disrupting cells.

Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.

III. Material and Methods

a. Plasmids Used

Negative control BBa_R0040 (provided in kit plate)
Positive control BBa_I20270 (provided in kit plate)
Test Device 1 BBa_J364000 (provided in kit plate)
Test Device 2 BBa_J364001 (provided in kit plate)
Test Device 3 BBa_J36400 (provided in kit plate)
Test Device 4 BBa_J364007 (provided in kit plate)
Test Device 5 BBa_J364008 (provided in kit plate)
Test Device 6 BBa_J364009 (provided in kit plate)

b. Strain used

  • Competent cells (Escherichia coli strain DH5α)
  • 1x PBS
  • ddH2O
  • LB media
  • Chloramphenicol
  • 50 ml Falcon tube
  • 1.5 ml Eppendorf tubes
  • Ice bucket with ice
  • Micropipettes
  • Micropipette tips
  • 96 well plates, black with clear flat bottom preferred
  • LUDOX CL-X (provided in kit)
  • Fluorescein sodium salt (provided in kit)
  • Silica Beads (Provided in kit)

    • c. Machines

    Spectrophotometer: Thermo scientific Varioskan Flash

    d. Software

    Microsoft Excel 2016

    f. Results

    OD600 Reference Point



    Table 1. OD600 Reference Point.

    Fluorescein Standard Curve






    Table2. Original data of Fluorescein standard curve

    Figure1. Fluorescein Standard Curve

    Particle Standard Curve

    Table 3. Original data of Particle Standard Curve

    Figure 2. Particle Standard Curve.

    Figure 3. Particle Standard Curve (log scale).

    Cell measurement

    Table 4. Fluorescence Intensity of E.coli transformed with Device 1 to 6 and two control groups. Intensities was measured under OD600 using Plate Reader for 6 hours.

    As shown in Table.4, the combination of RBS and promoter in Device exhibited the strongest fluorescent intensity in 6h. Among these six devices, Device 3 almost had no fluorescent intensity.

    Original data link:

    Table 5. Bacterial concentration of E.coli transformed with Device1-6, Negative Control & Positive Control. Intensities were measured under OD600 using plate for 6 hours.

    As shown in Table.5, except Device 1, the concentrations of E.coli transformed with another five devices were almost the same.

    Normalizing

    Counting colony-forming units

    Figure 5. Counted by Promega Colony Counter, bacterial concentration of E.coli transformed with Positive Control & Negative Control.

    As shown in Figure.5, bacterial colonies was counted in order to determine whether absolute cell count or colony-forming units (CFUs) can reduce lab-to-lab variability in fluorescence measurements.

    It is evident that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3. Additionally, CFUs cannot replace the lab measurement because bacterial colonies is so crowded that it is impossible to count precisely by Promega Colony Counter.

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