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

Revision as of 16:08, 16 October 2018

iGem HKUST 2018 Hielo by TEMPLATED

INTERLAB

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:

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
Test Device 1 (BBa_J364000)	Plate 7 Well 2F	BBa_J23101 (1791au)	BBa_B0034 (1.0)
Test Device 2 (BBa_J364001)	Plate 7 Well 2H	BBa_J23106 (1185au)
Test Device 3 (BBa_J364002)	Plate 7 Well 2J	BBa_J23117 (162au)
Test Device 4 (BBa_J364007)	Plate 7 Well 2L	BBa_J23100(2547au)	BBa_B0034* (nil)
Test Device 4 (BBa_J364007)	Plate 7 Well 2L	BBa_J23100(2547au)	BBa_B0034* (nil)

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

Table2: Conversion factor calculation

Responsive image — Fig. 2a Particle Standard Curve

Converting between 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:

Overall, the result obtained was reasonable. Among all device, device 1 reach overall highest fluorescence level while device show the lowest, the outcome is due to the different of promoter of GFP. Since the strength of promoter of device affect the expression of GFP. According to the Part Registry, promoter strength of device 1 (BBa_J23101), device 2 (BBa_J23106), device 3 (BBa_J23117), device 4 (BBa_J23100), device 5 (BBa_J23104) and device 6 (BBa_J23116) are 1791, 1185, 162, 2547, 1830 and 396 au respectively. This may explain the difference levels of GFP produced.

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

@@ Line 75: / Line 75: @@
 <ul>
 <h3><i>Materials:</i></h3><br/>
-<li><b>LUDOX CL-X,/b>: 45% colloidal silica suspension, used as single reference point for converting absorbance (Abs<sub>600</sub>) to OD<sub>600</sub> <br/>.
+<li><b>LUDOX CL-X</b>: 45% colloidal silica suspension, used as single reference point for converting absorbance (Abs<sub>600</sub>) to OD<sub>600</sub> <br/>.
 </li>
 <li><b>Silica beads</b>: Microsphere suspension that mimics the shape and size of typical <i>E.coli</i> cell.  With known concentration, it can be used for the conversion of absorbance measurement to the universal standard concentration of bead measurement.