Difference between revisions of "Team:Hong Kong HKUST/InterLab"

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

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

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 consistent value of cell concentration.

REFERENCES:

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

2. InterLab Plate Reader Protocol. The 2018 International Genetically Engineered Machine. Available:https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf