Difference between revisions of "Team:UiOslo Norway/InterLab"

InterLab

Once again, the iGEM foundation arranges the InterLab study and invited the teams to participate. This year, the study is centered around GFP-measurement. Specifically, testing the accuracy of GFP intensity measurement by the use of a plate reader, and comparing the data between the teams. Each team was given 8 GFP probes to transform DH5-alpha E. Coli-cells with, and were to measure the cell concentration and GFP excitation intensity of these, and send them in for comparison. The goal was to create a common protocol for GFP measurement in plate readers, by seeing how close the different teams would be able to get their results. At the end of the page there is an Excel document containing all results and calculations throughout fire sheets. During this interlab study we were given an interlab measurement kit providing us with plasmids listed in Table 1: Measurement Kit.

Table 1: Measurement Kit

Name	Marked on plate	Containing
Positive control	PC	-
Negative control	NC	-
TestDevice 1	T1	J23101+I13504
TestDevice 2	T2	J23106+I13504
TestDevice 3	T3	J23117+I13504
TestDevice 4	T4	J23101.BCD2.E0040.B0015
TestDevice 5	T5	J23106.BCD2.E0040.B0015
TestDevice 6	T6	J23117.BCD2.E0040.B0015

Protocol used is the E.coli DH5Alpha protocol provided form iGEM. Step 15 and 16 was shipped since colony growth indicates successful transformation. Pictures of the plates from step 13 in the protocol are shown below.

Pictures of the plates with colonies:

• Positive control
• Negative Control
• Probe T1
• Probe T2
• Probe T3
• Probe T4
• Probe T5
• Probe T6

The overnight(ON) cultures wore made as specified in the protocol provided form iGEM. Furthermore, the cultures wore diluted to OD600 = 0.002 using OD reader and dilution calculation Sheet_1 as specified. Altho the notes wore misplaced and the initial OD for the ON cultures is unknown. However, looking at Table 2: OD600 measurements for 0h. below, we see that the OD600 is closer to 0.004-0.005 than 0.002. This indicates a measurement or pipetting error for our part. Altho starting OD is wrong, the increase in OD and FI wore prosiding as expected (Sheet: Raw Plate Reader Measurements).

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.045	0.047	0.045	0.045	0.046	0.048	0.048	0.048	0.044
Colony 1, Replicate 2	0.047	0.047	0.047	0.05	0.044	0.049	0.047	0.048	0.042
Colony 1, Replicate 3	0.047	0.046	0.047	0.046	0.042	0.048	0.049	0.048	0.045
Colony 1, Replicate 4	0.047	0.047	0.052	0.047	0.043	0.046	0.047	0.048	0.043
Colony 2, Replicate 1	0.051	0.053	0.048	0.049	0.059	0.048	0.049	0.054	0.045
Colony 2, Replicate 2	0.046	0.048	0.046	0.047	0.048	0.048	0.049	0.048	0.044
Colony 2, Replicate 3	0.05	0.047	0.048	0.045	0.043	0.047	0.046	0.045	0.043
Colony 2, Replicate 4	0.048	0.048	0.048	0.048	0.047	0.048	0.047	0.048	0.044

More changes to the iGEM protocol wore made. Since we did not have 96 well plates, black with flat, transparent/clear bottom we used 96 well plate, black with flat black bottom for FI measuring and 96 well plate, transparent with flat transparent bottom for the OD600 measuring. This setup was also used for the standard curves. To have enough sample for two plates instead of one, we had to take 1 mL samples and not 500 uL. Even though we used two plate, the cultures wore the same, and OD corresponds to FI. Furthermore, looking at the Excel: Raw Plate Reader Measurements we see that the positive control increases i OD and FI, while the negative control increases in OD, but not FI.

Link to the sheet