Team:Chalmers-Gothenburg/InterLab

Interlab - iGEM Chalmers-Gothenburg 2018

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InterLab

We participated in iGEMs fifth InterLab Study, an international collaboration where teams from all over the world participates. The study aims at establishing new reliable tools for the iGEM community and for synthetic biology as a whole. One of the staples of synthetic biology is green fluorescence protein (GFP), but fluorescence data from this marker can be hard to compare as it can be reported using different units or processed in different ways. The InterLab study aims to establish detailed protocols and a data analysis form which can be used to yield absolute units for the fluorescence measurements. This year’s study aims at reducing the variability of having different amount of cells in the samples by normalising to colony forming units (CFU) instead of optical density (OD).

Measurement of LUDOX CL-X OD600 Reference Point

By measuring the absorbance of LUDOX CL-X silica beads and comparing it with a reference OD, we can find the conversion factor used for converting the absorbance measurements from our plate reader to standardised OD measurements.

Table 1: LUDOX CL-X absorbance measurements and the resulting convertion factor
LUDOX CL-X H2O
Replicate 1 0,084 0,059
Replicate 2 0,059 0,072
Replicate 3 0,056 0,078
Replicate 4 0,086 0,002
Arith. Mean 0,071 0,053
Corrected Abs600 0,019
Reference OD600 0,063
OD600/Abs600 3,405

Particle Standard Curve

In order to convert our absorbance measurements to an estimated amount of cells a concentration curve is needed. By using a known amount silica beads with similar characteristics as E.coli cells, a standard curve can be created.

Table 2: Data from silica beads absorbance readings
Number of particles 2,35E+08 1,18E+08 5,88E+07 2,94E+07 1,47E+07 7,35E+06 3,68E+06 1,84E+06 9,19E+05 4,60E+05 2,30E+05 0
Replicate 1 0,839 0,39 0,226 0,088 0,01 0,005 0,007 0,021 0,008 0,013 0,023 0,035
Replicate 2 0,874 0,444 0,217 0,126 0,092 0,024 0,011 0,014 -0,002 0,0001 0,013 0,028
Replicate 3 0,821 0,419 0,256 0,122 0,061 0,026 0,086 0,033 0,011 0,043 -0,022 0,033
Replicate 4 0,872 0,417 0,254 0,136 0,079 0,05 0,032 0,016 0,03 0,018 0,016 0,053
Arith. Mean 0,852 0,418 0,238 0,118 0,061 0,026 0,034 0,021 0,012 0,019 0,008 0,037
Arith. Std.Dev. 0,026 0,022 0,020 0,021 0,036 0,018 0,036 0,009 0,013 0,018 0,020 0,011
Arith. Net Mean 0,814 0,380 0,201 0,081 0,023 -0,011 -0,003 -0,016 -0,026 -0,019 -0,030
Particle standard curve
Figure 1: Particle standard curve
Particle logarithmic standard curve
Figure 2: Particle logarithmic standard curve

Fluorescence Standard Curve

Fluorescence values vary a lot between instruments. A standard curve can be created using known expressions of fluorescein protein. The values from our GFP measurements can then be converted into a corresponding fluorescein concentration.

Table 3: Flourescein flourescence readings
Flourescein µM 10,00 5 2,5 1,25 0,625 0,313 0,156 0,078 0,039 0,0195 0,0098 0
Replicate 1 13172 7135 3785 1965 977 494 259 145 80 50 39 21
Replicate 2 14283 7199 3707 1872 973 496 262 139 79 50 36 23
Replicate 3 14274 7264 3735 1915 989 499 271 147 83 53 36 19
Replicate 4 14360 7429 3618 1888 965 494 264 142 80 51 38 22
Arith. Mean 14020 7257 3711 1910 976 495,8 264 143,3 80,5 51 37,25 21,25
Arith. Std.Dev. 568,1 126,3 70,04 40,73 10 2,363 5,099 3,5 1,732 1,414 1,5 1,708
Arith. Net Mean 14000 7236 3690 1889 954,8 474,5 242,8 122 59,25 29,75 16
Flourescein standard curve
Figure 3: Flourescein standard curve
Flourescein logarithmic standard curve
Figure 4: Florescein standard logarithmic curve

Transformation, Cell Growth, and Measurements

Six different constructs coding for GFP were transformed into E.coli. Colonies from these were grown overnight in liquid media before being diluted to a set optical density. The cells were left to incubate for 6 hours, with sampling taking place after 0 and 6 hours. These samples were analysed for absorbance and fluorescence.

Table 4: Flourescence readings after 0 hours
Flourescence 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 1447 1706 4064 2339 1482 4967 5452 2143 1385
Colony-1, Replicate-2 1425 1694 3870 2179 1469 4736 5507 2029 1471
Colony-1, Replicate-3 1513 1742 4204 2309 1457 4760 5504 2083 1420
Colony-1, Replicate-4 1529 1619 4154 2248 664 4954 5263 2045 1506
Colony-2, Replicate-1 1566 1898 4689 2225 1584 2591 4631 1889 1443
Colony-2, Replicate-2 1653 1856 4644 2322 1521 2668 4826 1867 1414
Colony-2, Replicate-3 1577 1905 4631 2284 1719 2644 4712 1780 1441
Colony-2, Replicate-4 1568 1926 4723 2349 1550 2520 4678 1858 1447
Table 5: Flourescence readings after 6 hours
Flourescence hour 6 Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony-1, Replicate-1 1913 13041 17199 24487 2340 71368 10878 12825 1235
Colony-1, Replicate-2 2031 14082 18594 26617 2195 74064 12977 13334 1282
Colony-1, Replicate-3 2299 14524 19614 26639 2361 77127 12440 14274 1304
Colony-1, Replicate-4 1993 15234 19551 26502 2280 75721 12581 13979 1421
Colony-2, Replicate-1 1884 15393 15355 25001 2047 42837 9181 13405 1281
Colony-2, Replicate-2 1978 17563 16377 25624 2176 45036 9232 13852 1326
Colony-2, Replicate-3 2083 16710 17034 26937 2203 47858 9176 13903 1281
Colony-2, Replicate-4 1943 16402 17010 26702 2108 45995 9009 14744 1296
Table 6: Absorbance readings after 0 hours
Absorbance 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 0,159 0,105 0,048 -0,004 0,024 0,03 0,038 0,073 0,082
Colony-1, Replicate-2 0,146 0,075 0,091 0,079 0,023 0,069 0,024 0,084 0,107
Colony-1, Replicate-3 0,049 0,102 0,129 0,125 0,035 0,093 0,073 0,109 0,087
Colony-1, Replicate-4 0,083 0,12 0,091 0,047 1,018 0,052 0,067 0,049 0,002
Colony-2, Replicate-1 0,044 0,077 0,017 0,045 0,005 0,06 0,108 0,099 0,049
Colony-2, Replicate-2 0,025 0,019 0,005 0,016 0,032 0,01 0,046 0,014 0,03
Colony-2, Replicate-3 0,047 0,023 0,008 0,014 0,025 0,029 0,06 0,01 0,044
Colony-2, Replicate-4 0,035 0,036 0,011 0,02 0,054 0,036 0,018 0,042 0,02
Table 7: Absorbance readings after 6 hours
Absorbance hour 6 Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony-1, Replicate-1 0,414 0,348 0,108 0,345 0,451 0,387 0,04 0,321 -0,003
Colony-1, Replicate-2 0,454 0,377 0,141 0,415 0,392 0,324 0,058 0,341 0,001
Colony-1, Replicate-3 0,46 0,383 0,138 0,417 0,434 0,366 0,06 0,398 0,004
Colony-1, Replicate-4 0,438 0,396 0,138 0,409 0,402 0,337 0,049 0,379 -0,003
Colony-2, Replicate-1 0,399 0,379 0,1 0,383 0,387 0,272 0,068 0,446 0,006
Colony-2, Replicate-2 0,44 0,425 0,118 0,41 0,426 0,322 0,085 0388 0,0008
Colony-2, Replicate-3 0,488 0,422 0,124 0,436 0,413 0,258 0,057 0,406 -0,0009
Colony-2, Replicate-4 0,424 0,422 0,108 0,415 0,38 0,279 0,035 0,412 -0,0004

Colony Forming Units

Some of the overnight culture was used to measure the colony forming units (CFU). The cultures were set to a specific OD and a serial dilution was performed for each sample and spread on 36 different plates. These plates were incubated overnight and the number of colonies present after 18 hours were counted, and the CFU/ml in the starting samples could be calculated.

Table 8: Colony count for serial dilutions
Dilution factor 8E+04 8E+05 8E+06
Pos. Ctrl. Col. 1 Replicate 1 >300 92 9
Pos. Ctrl. Col. 1 Replicate 2 >300 134 12
Pos. Ctrl. Col. 1 Replicate 3 >300 151 5
Pos. Ctrl. Col. 2 Replicate 1 >300 221 25
Pos. Ctrl. Col. 2 Replicate 2 >300 154 15
Pos. Ctrl. Col. 2 Replicate 3 >300 148 15
Neg. Ctrl. Col. 1 Replicate 1 >300 176 14
Neg. Ctrl. Col. 1 Replicate 2 >300 165 21
Neg. Ctrl. Col. 1 Replicate 3 >300 165 19
Neg. Ctrl. Col. 2 Replicate 1 >300 161 52
Neg. Ctrl. Col. 2 Replicate 2 >300 153 72
Neg. Ctrl. Col. 2 Replicate 3 >300 135 56
Table 9: CFU for serial dilutions
Dilution factor 8E+04 8E+05 8E+06
Pos. Ctrl. Col. 1 Replicate 1 NA 7,4E+07 7,2E+07
Pos. Ctrl. Col. 1 Replicate 2 NA 1,1E+08 9,6E+07
Pos. Ctrl. Col. 1 Replicate 3 NA 1,2E+08 4E+07
Pos. Ctrl. Col. 2 Replicate 1 NA 1,8E+08 2E+08
Pos. Ctrl. Col. 2 Replicate 2 NA 1,2E+08 1,2E+08
Pos. Ctrl. Col. 2 Replicate 3 NA 1,2E+08 1,2E+08
Neg. Ctrl. Col. 1 Replicate 1 NA 1,4E+08 1,1E+08
Neg. Ctrl. Col. 1 Replicate 2 NA 1,3E+08 1,7E+08
Neg. Ctrl. Col. 1 Replicate 3 NA 1,1E+08 1,5E+08
Neg. Ctrl. Col. 2 Replicate 1 NA 1,3E+08 4,2E+08
Neg. Ctrl. Col. 2 Replicate 2 NA 1,2E+08 5,8E+08
Neg. Ctrl. Col. 2 Replicate 3 NA 1,1E+08 4,5E+08

Lab Journal

Interlab Lab Journal