Team:TUST China/InterLab

nav

inter

Interlab

We would like to thank all of those who have supported and assisted us in the laboratory, in the school and in society since the beginning of our tetracycline detecting and degradation project. Without those help, this project wouldn't have been possible and cannot been progressing well.

Introduction

Fluorescence data usually cannot be compared because it has been reported in different units or because different groups process data in different ways. Many have tried to work around this using “relative expression” comparisons; however, being unable to directly compare measurements makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.

The InterLab protocol aims to address these issues by providing researchers with a detailed protocol and data analysis form that yields absolute units for measurements of GFP in a plate reader.

Instrumentation

We used SpectraMax i3x+MinMax to measure Abs600 and fluorescent values. All the samples were taken from the distribution kit. The instrument settings were kept as: Excitation/Emission wavelengths-485nm/525nm, Bandwidth-25. Abs600 sample measurements were taken in 96 well micro test plate.

Experimental Design

  • For this study，first of all, we measured the Abs600 of LUDOX and MiliQ, the Abs600 of diluted monodisperse silica microspheres and the fluorescence of 1x fluorescein with the plate reader according to the InterLab Study Protocol.

      • Table 1:The Abs600 of LUDOX and MiliQ
        LUDOX CL-X H2O
        Replicate 1 0.0591 0.0419
        Replicate 2 0.0555 0.0438
        Replicate 3 0.0543 0.0449
        Replicate 4 0.0575 0.0391
        Arith.Mean 0.057 0.052
        Corrected Abs600 0.014  
        Reference OD600 0.063  
        OD600/Abs600 4.444  


        Table 2: The Abs600 of Diluted Monodisperse Silica Microspheres
        Number of Particle 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
        0Replicate 1 0.735 0.426 0.271 0.188 0.105 0.075 0.054 0.052 0.040 0.040 0.039 0.042
        Replicate 2 0.719 0.411 0.249 0.141 0.100 0.063 0.053 0.043 0.039 0.038 0.037 0.039
        Replicate 3 0.787 0.475 0.268 0.183 0.114 0.072 0.053 0.048 0.041 0.040 0.039 0.040
        Replicate 4 0.719 0.432 0.259 0.146 0.102 0.078 0.054 0.042 0.039 0.038 0.038 0.037
        Arith.Mean 0.740 0.436 0.262 0.164 0.105 0.072 0.054 0.046 0.040 0.039 0.038 0.040
        Arith.std.Dev 0.032 0.027 0.010 0.024 0.006 0.007 0.000 0.005 0.001 0.001 0.002 0.002
        Arith.Net.Mean 0.700 0.396 0.222 0.125 0.066 0.032 0.014 0.007 0.000 -0.01 -0.02  


        Figure 1: Particle Standard Curve


        Table 3:The Fluorescence of Fluorescein in PBS for Different Concentrations (µM)
        Flourescein uM 10.00 5 2.5 1.25 0.625 0.313 0.156 0.078 0.039 0.0195 0.0098 0
        Replicate1 370606592 161926416 100917072 53663002 23742310 12214789 6448555 3407568 1788076 900426 537111 101432
        Replicate2 345441472 170366720 92624232 50709240 28655780 11559271 6388769 3493244 1517289 843113 443478 105500
        Replicate3 353866656 197401504 105190296 47617436 22725132 12143993 5677301 3132938 1465665 887767 412216 100266
        Replicate4 366541504 175764736 120099512 49659592 25670368 11558932 6590084 2315652 1571070 927633 472234 110210
        Arith.Mean 3.591×108 1.764×108 1.047×108 5.041×107 2.520×107 1.187×107 6.276×106 3.085×106 1.586×106 8.897×105 4.663×105 1.044×105
        Arith.std.Dev 1.157×107 1.514×107 1.151×107 2.519×106 2.609×106 3.593×105 4.081×105 5.366×105 1.417×105 3.525×105 5.321×105 1.044×105
        Arith.Net.Mean 3.590×108 1.763×108 1.046×108 5.031×107 2.509×107 1.176×107 6.176×106 2.980×106 1.481×106 7.854×105 3.619×105  




        Figure 2: Fluorescein Standard Curve

        With the LUDOX measurement we were able to convert the measured Abs600 to OD600 with a reference OD600. With the Monodisperse Silica Microspheres measurement we were able to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells. With the fluorescence of fluorescein we were able to create a standard curve to be able to convert the fluorescence of the cells into a concentration of GFP.

  • After that, we measured six different test device, a negative and a positive control.

  • We transformed the eight different plasmids into Escherichia coli strain DH5α. The positive control is a plasmid that consists of a promoter and GFP (BBa_I20270) in the pSB1C3 backbone, and the negative control a plasmid that consists of a TetR repressible promoter (Bba_R0040) in the pSB1C3 backbone.The plasmids of the six test devices in the pSB1C3 backbone consist of a constitutive GFP expressing promoter with different intensities of expression effects. These plasmid were delivered to us with the iGEM distribution kit.

  • From the transformation plates we picked nine colonies of every plate and did a colony PCR and gel electrophoresis.

  • We then picked two colonies of each plate that had the insert, and incubate them in liquid LB at 37℃ and 220rpm for 16-18 hours.(16 tubes totally)

  • Dilute and smear plate of negative and positive colonies.（36 plates totally)

    • Culture them at 44℃ for one night.

  • After diluting we took 500µL samples out of every tube into 1.5 ml eppendorf tubes, and placed the samples on ice.

  • After that, we incubated the remainder of the cultures at 37°C and 220 rpm for 6 hours.

  • Then, we measured the Abs600 and fluorescence of these samples.

      • Table 4:The Fluorescence of Plasmids at 0h and 6h
        Hour0: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony1,Replicate 1228466 7635254 2057614 1304663 7744215 9130150 1826135 1920430 911447
        Colony1,Replicate 1251196 8353847 2075158 1402286 7450288 9179034 1841801 1954181 1008752
        Colony1,Replicate 1261807 9743852 2108677 1269841 6943626 9988806 1847625 1892470 1090781
        Colony1,Replicate 1208369 8028170 2035486 1274315 7253590 8323066 1782061 1772216 888156
        Colony2,Replicate 1180019 6792732 1852742 1213931 8899422 18735560 1894645 1978050 956476
        Colony2,Replicate 1200042 6435040 1918579 1346352 8341680 17974298 1880017 2119853 951358
        Colony2,Replicate 1236813 6308314 1812273 1348795 9302734 17714382 1785925 2001931 965727
        Colony2,Replicate 1215422 9894252 1966804 1302884 8704658 18278350 1999645 2081853 998718
        Hour6: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony1,Replicate 1086166 14243759 1526318 1253152 19263920 10491237 4107560 3393461 624592
        Colony1,Replicate 1082993 13749219 1485822 1284858 19311876 9155288 3977902 3418002 640187
        Colony1,Replicate 1019804 14558605 1455743 1202266 19204552 9213459 3817949 3166506 622100
        Colony1,Replicate 1061223 13919501 1369135 1120664 18808648 9568944 3745423 3029380 576213
        Colony2,Replicate 1177375 16315975 1388384 1506507 17541648 15825251 3781017 4967098 586023
        Colony2,Replicate 1128354 14427503 1412517 1314042 21950936 16294213 3819550 3541690 585576
        Colony2,Replicate 1229430 15774420 1330589 1300820 18055844 15794978 4104556 4346300 551686
        Colony2,Replicate 1212704 16428246 1418167 1384998 17152636 15774047 3779394 4102795 568564



        Figure 3: Contrast The Fluorescence of Plasmids at 0h and 6h


        Table 5: The Abs600 of Plasmids at 0h and 6h
        Hour0: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony 1, Replicate 1 0.1282 0.1658 0.1538 0.1457 0.1412 0.1148 0.1141 0.1373 0.0351
        Colony 1, Replicate 2 0.1214 0.1799 0.1498 0.1522 0.1344 0.1052 0.108 0.1168 0.0363
        Colony 1, Replicate 3 0.1234 0.219 0.1542 0.1417 0.1309 0.1073 0.1068 0.1197 0.041
        Colony 1, Replicate 4 0.1291 0.1828 0.1517 0.1367 0.1261 0.1074 0.109 0.1151 0.0385
        Colony 2, Replicate 1 0.1702 0.1576 0.1596 0.1468 0.202 0.1135 0.133 0.1603 0.0355
        Colony 2, Replicate 2 0.137 0.1418 0.1551 0.1327 0.1958 0.1096 0.125 0.1436 0.0369
        Colony 2, Replicate 3 0.2035 0.1416 0.1584 0.1362 0.1999 0.1066 0.1101 0.1234 0.0344
        Colony 2, Replicate 4 0.1189 0.2285 0.1539 0.1295 0.1903 0.1067 0.115 0.1198 0.0346
        Hour6: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony 1, Replicate 1 0.4958 0.57 0.5815 0.5435 0.5928 0.5943 0.54 0.5447 0.0368
        Colony 1, Replicate 2 0.5143 0.5522 0.628 0.5484 0.5922 0.6039 0.531 0.5441 0.0376
        Colony 1, Replicate 3 0.5095 0.57 0.6502 0.5385 0.584 0.5849 0.5207 0.5099 0.0375
        Colony 1, Replicate 4 0.5153 0.5531 0.5063 0.5292 0.5908 0.5887 0.5235 0.5186 0.0359
        Colony 2, Replicate 1 0.8429 0.6241 0.6023 0.6101 0.5535 0.5915 0.5193 0.6119 0.039
        Colony 2, Replicate 2 0.8312 0.6016 0.517 0.5764 0.5622 0.5631 0.5046 0.4888 0.0413
        Colony 2, Replicate 3 0.8335 0.6186 0.5714 0.5711 0.5504 0.5646 0.5108 0.5749 0.0377
        Colony 2, Replicate 4 0.8202 0.6141 0.7328 0.5738 0.535 0.5708 0.5011 0.5603 0.0359




        Figure 4: Contrast The Abs600 of Plasmids at 0h and 6h



        Table 6: Fluorescence Per OD
        Hour0: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony 1, Replicate 1 0.0197 0.2977 0.0559 0.0206 0.3726 0.5967 0.0670 0.0571
        Colony 1, Replicate 1 0.0165 0.2960 0.0544 0.0196 0.3799 0.6861 0.0672 0.0680
        Colony 1, Replicate 1 0.0120 0.2813 0.0520 0.0103 0.3767 0.7766 0.0666 0.0589
        Colony 1, Replicate 1 0.0205 0.2863 0.0586 0.0228 0.4205 0.6244 0.0734 0.0668
        Colony 2, Replicate 1 0.0096 0.2766 0.0418 0.0134 0.2760 1.3549 0.0610 0.0634
        Colony 2, Replicate 1 0.0144 0.3025 0.0473 0.0239 0.2691 1.3549 0.0627 0.0634
        Colony 2, Replicate 1 0.0093 0.2884 0.0395 0.0218 0.2915 1.3423 0.0627 0.0674
        Colony 2, Replicate 1 0.0149 0.2655 0.0470 0.0185 0.2864 1.3867 0.0720 0.0736
        Hour6: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony 1, Replicate 1 0.4958 0.57 0.5815 0.5435 0.5928 0.5943 0.54 0.5447 0.0368
        Colony 1, Replicate 2 0.5143 0.5522 0.628 0.5484 0.5922 0.6039 0.531 0.5441 0.0376
        Colony 1, Replicate 3 0.5095 0.57 0.6502 0.5385 0.584 0.5849 0.5207 0.5099 0.0375
        Colony 1, Replicate 4 0.5153 0.5531 0.5063 0.5292 0.5908 0.5887 0.5235 0.5186 0.0359
        Colony 2, Replicate 1 0.8429 0.6241 0.6023 0.6101 0.5535 0.5915 0.5193 0.6119 0.039
        Colony 2, Replicate 2 0.8312 0.6016 0.517 0.5764 0.5622 0.5631 0.5046 0.4888 0.0413
        Colony 2, Replicate 3 0.8335 0.6186 0.5714 0.5711 0.5504 0.5646 0.5108 0.5749 0.0377
        Colony 2, Replicate 4 0.8202 0.6141 0.7328 0.5738 0.535 0.5708 0.5011 0.5603 0.0359




        Table7: Fluorescence Per Particle
        Hour0: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony 1,Replicate 1 2.11E+03 3.19E+04 5.99E+03 2.21E+03 3.99E+04 6.40E+04 7.18E+03 6.12E+03
        Colony 1,Replicate 2 1.77E+03 3.17E+04 5.83E+03 2.11E+03 4.07E+04 7.36E+04 7.21E+03 7.28E+03
        Colony 1,Replicate 3 1.29E+03 3.02E+04 5.58E+03 1.10E+03 4.04E+04 8.32E+04 7.13E+03 6.32E+03
        Colony 1,Replicate 4 2.19E+03 3.07E+04 6.29E+03 2.44E+03 4.51E+04 6.69E+04 7.86E+03 7.16E+03
        Colony 2,Replicate 1 1.03E+03 2.96E+04 4,48E+03 1.43E+03 2.96E+04 1.41E+05 5.97E+03 5.08E+03
        Colony 2,Replicate 2 1.54E+03 3.24E+04 5.08E+03 2.56E+03 2.88E+04 1.45E+05 6.54E+03 6.79E+03
        Colony 2,Replicate 3 9.94E+02 3.09E+04 4.23E+03 2.33E+03 3.12E+04 1.44E+05 6.72E+03 7.22E+03
        Colony 2,Replicate 4 1.59E03 2.85E04 5.03E03 1.99E03 3.07E04 1.49E05 7.72E03 7.89E03
        Hour6: Neg.Control Pos.Control Device2 Device3 Device4 Device5 Device6 LB+Chlor(blank)
        Colony 1,Replicate 1 6.24E+02 1.58E+04 1.03E+03 7.69E+02 2.08E+04 1.10E+04 4.29E+03 3.38E+03
        Colony 1,Replicate 2 5.76E+02 1.58E+04 8.88E+02 7.83E+02 2.09E+04 9.33E+03 4.20E+03 3.40E+03
        Colony 1,Replicate 3 5.23E+02 1.62E+04 8.44E+02 7.18E+02 2.11E+04 9.74E+03 4.10E+03 3.34E+03
        Colony 1,Replicate 4 6.28E+02 1.60E+04 1.05E+03 6.85E+02 2.04E+04 1.01E+04 4.03E+03 3.15E+03
        Colony 2,Replicate 1 4.56E+02 1.67E+04 8.84E+02 1.00E+03 2.04E+04 1.71E+04 4.13E+03 4.74E+03
        Colony 2,Replicate 2 4.26E+02 1.53E+04 1.08E+03 8.44E+02 2.54E+04 1.87E+04 4.33E+03 4.10E+03
        Colony 2,Replicate 3 5.28E+02 1.63E+04 9.05E+02 8.71E+02 2.12E+04 1.79E+04 4.66E+03 4.38E+03
        Colony 2,Replicate 4 5.09E+02 1.70E+04 7.56E+02 9.41E+02 2.06E+04 1.76E+04 4.28E+03 4.18E+03


  • Plate reading.

      • Table 8: The Number of Colonies for Different Concentrations
        8×10-4 8×10-5 8×10-6
        B1-1 1210 130 12
        B1-2 1143 123 13
        B1-3 1120 102 15
        B2-1 1334 150 12
        B2-2 1012 92 10
        B2-3 972 88 10
        D1-1 1173 144 14
        D1-2 1090 106 13
        D1-3 1202 116 14
        D2-1 1045 99 9
        D2-2 1187 121 14
        D2-3 996 90 7


        Table 9: Colony Forming Units per 0.1 OD600 E. coli cultures
        8×105 9×106
        B1-1 1.04×108 9.6×107
        B1-2 9.84×107 1.04×108
        B1-3 8.16×107 1.20×108
        B2-1 1.20×108 9.60×107
        B2-2 7.36×107 8.00×107
        B2-3 7.04×107 8.00×107
        D1-1 1.152×108 1.128×108
        D1-2 8.48×107 1.048×108
        D1-3 9.28×107 1.128×108
        D2-1 7.92×107 7.20×107
        D2-2 9.68×107 1.128×108
        D2-3 7.20×107 5.60×107


    Conclusions

    The higher expression of the fluorescence, the stronger of the promoter. The strength of the promoter also affects the growth of the cells: the growth rate of high promoter is slow, which can be seen from the figure 3 and 4. Test device 3 showed the highest fluorescence expression during growth, while it grew a bit of slower than others. All the devices grew well, but only device 3, 5 and 6 can express fluorescence efficiently.

    What’s more, the fluorescence per OD and the fluorescence per particle of all the devices decreased according to the data from the table 6 and 7. That is to say, with the increase of cell concentration, the total amount of fluorescence increased, but the fluorescence volume per unit cell decreased.

    foot