Difference between revisions of "Team:Aix-Marseille/InterLab"

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Revision as of 16:57, 11 October 2017

InterLab

How close can the numbers be when fluorescence is measured all around the world ?

In order to support the interlab approach, our team participate to the Fourth International InterLab Measurement Study. We are glad to contribute to this studie based on reliable and repeatable measurement. The challenge this year consist in etablishing a GFP measurement protocol that can be used produce comparable GFP measurements on plate readers. Furthermore, teams are also going to test some RBS devices that are intended to make gene expression more precise and reliable.


Fluorescence of GFP of E.Coli


Materials and Methods:

Used plasmids

Devices from Interlab Measurement Kit

  1. Positive control
  2. Negative control
  3. Test Device 1:J23101.BCD2.E0040.B0015
  4. Test Device 2:J23106.BCD2.E0040.B0015
  5. Test Device 3:J23117.BCD2.E0040.B0015
  6. Test Device 4:J23101+I13504
  7. Test Device 5:J23106+I13504
  8. Test Device 6:J23117+I13504

Used strain

E. coli E5alpha

Used material

1ml LUDOX H20 96 well plate,black with flat bottom fluorescein 10ml 1xPBS LB (Luria Bertani) media Chloramphenicol (stock concentration 25mg/mL dissolved in EtOh - working stock 25 ug/ml) 50 ml Falcon tube Incubator at 37°C 1.5 ml eppendorf tube Ice bucket Pipettes


Used machines

  1. Tecan's Infinite®
  2. Confocal microscope

Used software


Link protocoles

T--Aix-Marseille--fluo interlab.jpeg



Description :

We used plasmids containing different promoter from the 2017 distribution kit and transfected them into E.coli. Before measuring fluorescence expression, we used LUDOX and fluorescein to calibrate the TECAN. We prepared a dilution series of fluorescein in 4 duplicates and measure the fluorescence in a 96 well plate in a plate reader. By measuring these in all standard modes, we generated a standard curve of fluorescence for fluorescein concentration. We used this to correct cell based readings to an equivalent fluorescein concentration and convert this into a concentration of GFP.



T--Aix-Marseille--fluoresceincurve.jpeg



Results


T--Aix-Marseille--fluorescencecourberesultats.jpeg



Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 156 165 167 177 157 159 153 153 147
Colony 1, Replicate 2 155 161 166 161 151 160 156 158 154
Colony 1, Replicate 3 158 157 164 152 144 159 151 155 147
Colony 1, Replicate 4 157 161 165 155 150 156 155 155 155
Colony 2, Replicate 1 158 159 163 178 154 158 158 153 152
Colony 2, Replicate 2 89 104 97 112 93 99 100 90 91
Colony 2, Replicate 3 94 103 99 109 93 103 99 97 89
Colony 2, Replicate 4 92 102 101 80 89 95 94 97 90


Hour 2: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 102 132 124 121 98 117 112 99 93
Colony 1, Replicate 2 101 126 120 125 99 109 118 96 93
Colony 1, Replicate 3 98 126 121 116 98 113 115 91 84
Colony 1, Replicate 4 99 128 122 118 91 111 115 93 92
Colony 2, Replicate 1 105 133 118 139 101 111 116 102 93
Colony 2, Replicate 2 99 128 113 139 103 110 114 104 95
Colony 2, Replicate 3 100 130 110 131 102 109 113 102 97
Colony 2, Replicate 4 96 132 116 133 101 116 112 99 97


Hour 4: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 109 228 168 250 105 147 152 105 93
Colony 1, Replicate 2 112 236 173 254 101 146 166 106 92
Colony 1, Replicate 3 112 243 174 259 101 154 162 108 85
Colony 1, Replicate 4 114 242 171 255 101 153 149 108 92
Colony 2, Replicate 1 103 224 157 262 99 151 156 106 102
Colony 2, Replicate 2 109 239 165 274 104 157 161 108 93
Colony 2, Replicate 3 110 239 161 263 105 162 159 109 91
Colony 2, Replicate 4 108 241 167 272 106 166 169 112 95


Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 148 362 348 481 147 343 211 142 120
Colony 1, Replicate 2 156 378 351 510 137 347 219 148 120
Colony 1, Replicate 3 150 382 318 497 138 355 218 153 120
Colony 1, Replicate 4 154 385 354 479 138 358 210 151 130
Colony 2, Replicate 1 149 354 336 493 136 375 210 150 130
Colony 2, Replicate 2 149 391 365 564 146 399 193 147 128
Colony 2, Replicate 3 154 367 355 507 140 416 210 149 130
Colony 2, Replicate 4 153 385 359 560 148 417 232 151 138