Difference between revisions of "Team:Hong Kong HKUST/InterLab"
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<center><figcaption><b>Fig.3a</b> Fluorescein standard curve</figcaption></center> | <center><figcaption><b>Fig.3a</b> Fluorescein standard curve</figcaption></center> | ||
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<h3>Conversion of absorbance of cells to absorbance of a known concentration of beads.</h3> | <h3>Conversion of absorbance of cells to absorbance of a known concentration of beads.</h3> | ||
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<center><figcaption><b>Fig.4a</b> Average <sub>u</sub>M Fluorescein / OD<sub>600</sub> of each devices | <center><figcaption><b>Fig.4a</b> Average <sub>u</sub>M Fluorescein / OD<sub>600</sub> of each devices | ||
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<center><figcaption><b>Fig.4b</b> Fluorescein standard curve (log scale) | <center><figcaption><b>Fig.4b</b> Fluorescein standard curve (log scale) | ||
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Revision as of 12:53, 17 October 2018
OUR INTERLAB OBJECTIVES
The iGEM Interlab 2018 aims to reduce lab-to-lab variability in fluorescence measurements that was shown in previous interlab studies to originate from using optical density (O.D.) as the normalisation method of fluorescence. Since O.D. is an approximation of cell number, the interlab this year attempts to address the problem by adopting a more direct normalisation method. That is, the use of absolute cell count or colony-forming units (CPU).
Method:
All procedures were performed according to the protocols iGEM given. 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:
Envision Multilabel Reader (Model: EnVision Xcite)
Machines:
*To know more about the setting of EnVision multilabel reader, please click
- 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
Materials:
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| 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 |
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.
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 |
| 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 |
| 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 |
- 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 normalised fluorescence values between using OD and particle count, as illustrated in Figure 4 and 5. The normalised fluorescence values of the devices are consistent with their respective promoter strengths, with device 1 (BBa_J23101) as the highest (i.e. 1791 a.u.) and device 3 (BBa_J23117) as the lowest (i.e. 162 a.u.). However, cell quantification by colony-forming units failed to reproduce the modelled cell concentration by silica beads. This may conclude that the method may not be able to reduce lab-to-lab variability.
REFERENCES:
The 2018 International Genetically Engineered Machine. (17 July, 2018). Tracks/Measurement/Interlab study/Plate Reader Protocol. Retrieved from https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
Registry of Standard Biological Parts (2018-04-17) Retrieved from http://parts.igem.org/assembly/plates.cgi?id=5641