Team:Hong Kong HKUST/InterLab

iGem HKUST 2018 Hielo by TEMPLATED
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OUR INTERLAB OBJECTIVES

The iGEM Interlab 2018 aims to reduce lab-to-lab variability in fluorescence measurements that were shown in previous interlab studies which use an optical density (O.D.) as the normalization method of fluorescence. Since O.D. is an approximation of cell number, the interlab this year attempts to address the problem by two orthogonal approaches. Hypothesized that silica beads have similar light scattering properties as the cells due to their similarities in size and shape, one of the approaches is to convert the absorbance of cells to the absorbance of a known concentration of silica beads [1]. Adopting a more direct normalization method, the other approach is to normalize the absorbance of cells by absolute cell counts or colony-forming units (CFU).

Method:

All procedures were performed according to the given iGEM protocol [2], except that the O.D. measurement setting was changed from OD600 to OD595, due to the limited options of plate reader in HKUST. After further discussion with the iGEM headquarter, we retained the data to be OD595.

Machines, materials and parts:

Machines:


  • Envision Multilabel Reader (Model: EnVision Xcite)


*To know more about the setting of EnVision multilabel reader, please click

Materials:


  • LUDOX CL-X: 45% colloidal silica suspension, used as single reference point for converting absorbance (Abs600) to OD600
    .
  • Silica beads: Microsphere suspension that mimics the shape and size of typical E.coli cell. With known concentration, it can be used for the conversion of absorbance measurement to the universal standard concentration of bead measurement.

  • Fluorescein: Sodium fluorescein was used for obtaining the standard fluorescence curve.

  • E.coli strain DH5αCompetent cell: used for transformation, the protocol used for making it can view in here

  • Parts:


    Parts Parts location on the kits plate Parts used as the promoter(strength) Parts used as the RBS(Efficiency) Reporter Gene Parts used as the Terminator
    Positive Control(BBa_I20270) Plate 7 Well 2B BBa_J23151 (nil) BBa_B0032 (0.3) GFP BBa_B0010, BBa_B0012
    Negative Control (BBa_R0040) Plate 7 Well 2D BBa_R0040 (nil) nil GFP BBa_B0010, BBa_B0012
    Test Device 1 (BBa_J364000) Plate 7 Well 2F BBa_J23101 (1791au) BBa_B0034 (1.0) GFP BBa_B0010, BBa_B0012
    Test Device 2 (BBa_J364001) Plate 7 Well 2H BBa_J23106 (1185au) BBa_B0034 (1.0) GFP BBa_B0010, BBa_B0012
    Test Device 3 (BBa_J364002) Plate 7 Well 2J BBa_J23117 (162au) BBa_B0034 (1.0) GFP BBa_B0010, BBa_B0012
    Test Device 4 (BBa_J364007) Plate 7 Well 2L BBa_J23100(2547au) BBa_B0034* (nil) GFP BBa_B0010, BBa_B0012
    Test Device 4 (BBa_J364007) Plate 7 Well 2L BBa_J23100(2547au) BBa_B0034* (nil) GFP BBa_B0010, BBa_B0012

    Result:

    Calibrations:

    Conversion factor of OD600(OD600/Abs600) = 3.036
    Table 2: Conversion factor calculation

    LUDOX CL-X H20
    Replicate 1 0.045 0.024
    Replicate 2 0.045 0.025
    Replicate 3 0.044 0.024
    Replicate 4 0.049 0.027
    Arithmethic mean 0.046 0.025
    Corrected Abs600 0.021
    Reference OD600 0.063
    OD600/Abs600 3.036

    Responsive image
    Fig. 2a Particle Standard Curve

    Responsive image
    Fig.2b Particle Standard Curve (log scale)

    Responsive image
    Fig.3a Fluorescein standard curve

    Responsive image
    Fig.3b Fluorescein standard curve (log scale)
    The non-linear fluorescence standard curve is conjectured to be a result of detector over-saturation.
    This could be inferred from a linear curve at low concentrations of fluorescein while reaching plateau at high concentrations.

    Conversion of absorbance of cells to absorbance of a known concentration of beads.


    Responsive image
    Fig.4a Average uM Fluorescein / OD600 of each devices

    Responsive image
    Fig.4b Fluorescein standard curve (log scale)

    Counting colony-forming units (CFUs) from the sample


    Colonies count:
    Negative control (BBa_R0040):

    Dillution 3 Dillution 4 Dillution 5
    Colony 1, Replicate 1 180 13 3
    Colony 1, Replicate 2 120 14 3
    Colony 1, Replicate 3 197 33 2
    Colony 2, Replicate 1 283 33 2
    Colony 2, Replicate 2 214 28 3
    Colony 2, Replicate 3 218 29 1

    Positive control ((BBa_I120270):

    Dillution 3 Dillution 4 Dillution 5
    Colony 1, Replicate 1 228 29 1
    Colony 1, Replicate 2 184 25 1
    Colony 1, Replicate 3 153 25 1
    Colony 2, Replicate 1 254 19 3
    Colony 2, Replicate 2 168 27 2
    Colony 2, Replicate 3 213 24 3

    Colony-forming unit (CFU): Negative control (BBa_R0040):

    Dillution 3 Dillution 4 Dillution 5
    Colony 1, Replicate 1 1.44E+07 1.04E+07 2.40E+07
    Colony 1, Replicate 2 9.60E+06 1.12E+07 2.40E+07
    Colony 1, Replicate 3 1.58E+07 2.64E+07 1.60E+07
    Colony 2, Replicate 1 2.26E+07 1.84E+07 1.60E+07
    Colony 2, Replicate 2 1.71E+07 2.24E+07 2.40E+07
    Colony 2, Replicate 3 1.74E+07 2.32E+07 8.00E+06

    Average

    • Colony 1: 1.69E+07 CFU/ml/0.1OD
    • Colony 2: 1.88E+07 CFU/ml/0.1OD
    • Average: 1.785E+07 CFU/ml/0.1OD
    • Using conversion factor OD/Abs= 3.036
    • Conversion factor: CFU/Abs/ml= 54.34 CFU/Abs/ml

    Conclusion:

    There is no significant difference in the pattern of normalized fluorescence values between using O.D. and particle count, as illustrated in Figure 4 and 5. The normalized fluorescence values of the devices are consistent with their respective promoter strengths, with device 1 (BBa_J23101) as the highest fluorescence value (i.e. 1791 a.u.) and device 3 (BBa_J23117) as the lowest fluorescence value (i.e. 162 a.u.). However, cell quantification by colony-forming units failed to reproduce the modeled cell concentration by silica beads. This may conclude that the two methods, CFU cell count and silica beads, may not be able to produce a consistent value of cell concentration.

    REFERENCES:

    1. Measurement/InterLab - 2018.igem.org", 2018.igem.org, 2018. Available: http://2018.igem.org/Measurement/InterLab

    2. InterLab Plate Reader Protocol. The 2018 International Genetically Engineered Machine. Available:http://2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf