Team:Kobe/InterLab


INTERLAB STUDY

We participated in the Fourth International InterLab Measurement Study. They asked us to measure the fluorescence of GFP in different devices in an attempt to explore the question, “How close can the numbers be when fluorescence is measured all around the world? “.

We followed the same protocols as directed in the iGEM protocol page.

Materials

E.coli DH5-alpha: Competent cells were purchased from TOYOBO
Plate reader: SH-9000Lab

Result

1. Calculation

Table 1. Abs600 of LUDOX-S40 and H2O
LUDOX-HS40 H2O
Replicate1 0.034 0.025
Replicate2 0.031 0.024
Replicate3 0.032 0.025
Replicate4 0.031 0.023
Arith.Mean 0.032 0.023
Corrected Abs600 0.00775
Reference OD600 0.0425
OD600/Abs600 5.483871

2. FIuorescein fluorescence standard curve

Table.2 shows the fluorescence measurement data, and Fig.1 shows the standard curve. We couldn’t measure the value of fluorescein fluorescence at 50, 25, 12.5, 6.25, 3.125 μM because they exceeded the measurable range.

Table 2. fluorescence data for fluorescein standard curve
uM Fluorescein 1.5625 0.78125 0.390625 0.195313 0.097656 0.048828 0
Replicate 1 57809 29980 15540 7861 3913 1949 3
Replicate 2 58424 30336 15369 7579 3948 1993 3
Replicate 3 58075 29993 15535 7723 3908 1999 2
Replicate 4 57953 29694 15532 7798 3935 2044 4
Arith. Mean 58065.25 30000.75 15494 7740.25 3926 1996.25 3
S.D. 262.7171 262.665 83.39864 121.4012 18.77942 38.86194 0.816497
Fig1. Fluorescein standard curve

3. Cell measurement

Table 3.Abs600 (cell concentration)
0h 2h 4h 6h
Negative Control (Colony 1) 0.00475 0.0275 0.14125 0.25875
Negative Control (Colony 2) 0.00975 0.0315 0.13275 0.235
Positive Control (Colony 1) 0.007 0.01475 0.12125 0.25875
Positive Control (Colony 2) 0.00825 0.019 0.1235 0.26925
Device 1 (Colony 1) 0.0045 0.00375 0.00875 0.01
Device 1 (Colony 2) 0.0045 0.00325 0.009 0.007
Device 2 (Colony 1) 0.006 0.01825 0.12825 0.2665
Device 2 (Colony 2) 0.00925 0.02325 0.141 0.26375
Device 3 (Colony 1) 0.0065 0.02225 0.13425 0.2735
Device 3 (Colony 2) 0.0105 0.0275 0.144 0.26325
Device 4 (Colony 1) 0.00375 0.00325 0.00875 0.0445
Device 4 (Colony 2) 0.0065 0.004 0.0085 0.0125
Device 5 (Colony 1) 0.00575 0.02375 0.14775 0.28175
Device 5 (Colony 2) 0.00875 0.019 0.1275 0.27675
Device 6 (Colony 1) 0.006 0.02425 0.1505 0.2855
Device 6 (Colony 2) 0.0085 0.02125 0.1005 0.2625
Fig.2: Abs600 (cell concentration)
Table 4: fluorescence
0h 2h 4h 6h
Negative Control (Colony 1) 39.5 26.25 123 79
Negative Control (Colony 2) 34.5 15 120 112
Positive Control (Colony 1) 72.5 316.25 1272.75 2534.25
Positive Control (Colony 2) 68.25 318.25 1269.25 2440.25
Device 1 (Colony 1) 557.5 563 940.5 1046.5
Device 1 (Colony 2) 461.75 355.25 633.75 781.25
Device 2 (Colony 1) 83.75 374.75 2280.25 5128.5
Device 2 (Colony 2) 116.25 494.25 2128.75 5528.5
Device 3 (Colony 1) 3 29.5 154.75 232.75
Device 3 (Colony 2) 28.5 29.75 161 258.5
Device 4 (Colony 1) 94 172.5 327.75 769
Device 4 (Colony 2) 71.75 165.5 286 443.5
Device 5 (Colony 1) 7.5 196 614.5 858.25
Device 5 (Colony 2) 14.5 189.75 796.75 987
Device 6 (Colony 1) 60.25 29.5 129.5 164
Device 6 (Colony 2) 33.75 26.5 97.75 165
Fig.3: Fluorescence
Table 6. Value of Flu/Abs600
0h 2h 4h 6h
Negative Control (Colony 1) 0.045689 0.00469 0.004282 0.001499
Negative Control (Colony 2) 0.018247 0.002376 0.004481 0.002352
Positive Control (Colony 1) 0.055533 0.105486 0.051542 0.048113
Positive Control (Colony 2) 0.04131 0.082437 0.050482 0.044579
Device 1 (Colony 1) 0.607923 0.745125 0.532061 0.520307
Device 1 (Colony 2) 0.522086 0.640948 0.355477 0.553925
Device 2 (Colony 1) 0.070761 0.101285 0.087415 0.094541
Device 2 (Colony 2) 0.063885 0.104249 0.074675 0.103012
Device 3 (Colony 1) 0.001716 0.006526 0.005688 0.004194
Device 3 (Colony 2) 0.01537 0.005312 0.005489 0.004827
Device 4 (Colony 1) 0.118182 0.265189 0.184017 0.084842
Device 4 (Colony 2) 0.054166 0.218167 0.166671 0.177319
Device 5 (Colony 1) 0.00281 0.040555 0.020409 0.014984
Device 5 (Colony 2) 0.007484 0.049747 0.030945 0.017517
Device 6 (Colony 1) 0.050096 0.005969 0.004225 0.002821
Device 6 (Colony 2) 0.024278 0.006149 0.004835 0.003078
Fig.4; Value of Flu/Abs600