Team:Jilin China/InterLab

INTERLAB


Interlab

  • Introduction

    Reliable and repeatable measurement remains one of the tough challenges in synthetic biology. In order to overcome this problem, the Measurement Committee, through the InterLab study, has been developing a measurement procedure for green fluorescent protein (GFP) for the last four years. The CFU count is a new feature of interlab study this year. The goal of the 2018 Interlab study is to determine how reliably known parts can conform to expectations and previous results and to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL. The CFU protocol assumes that 1 bacterial cell will give rise to 1 colony.

    For a more detailed description, please visit the homepage of the iGEM's measurement committee.

  • Device We Received

    Negative Control BBa_R0040
    Positive Control BBa_I20270
    Test Device 1 BBa_J364000
    Test Device 2 BBa_J364001
    Test Device 3 BBa_J364002
    Test Device 4 BBa_J364003
    Test Device 5 BBa_J364004
    Test Device 6 BBa_J364005

  • Protocol

    We strictly followed the protocol provided by iGEM. We measured the experiment of OD600 reference point and fitted the fluorescence standard curve first and then detected GFP expression in E.coli with the plate reader Synergy HT from BioTek.

    This is the transformation protocol.

    This is the interlab plate reader protocol.

  • Raw Data

    Table 1. The data of the OD600 reference point experiment
      LUDOX CL-X H2O
    Replicate 1 0.057 0.038
    Replicate 2 0.057 0.039
    Replicate 3 0.057 0.039
    Replicate 4 0.057 0.041
    Arith. Mean 0.057 0.039
    Corrected Abs600 0.018  
    Reference OD600 0.063  
    OD600/Abs600 3.549  
    Table 2. Particle standard curve experiment
    Number of Particles 235294117.647 117647058.824 58823529.412 29411764.706 14705882.353 7352941.176 3676470.588 1838235.294 919117.647 459558.824 229779.412 0.0
    Replicate 1 0.703 0.369 0.199 0.141 0.1 0.066 0.051 0.044 0.043 0.041 0.041 0.039
    Replicate 2 0.644 0.375 0.219 0.12 0.084 0.064 0.053 0.044 0.041 0.038 0.04 0.039
    Replicate 3 0.638 0.359 0.209 0.122 0.08 0.059 0.048 0.044 0.042 0.041 0.043 0.04
    Replicate 4 0.651 0.365 0.204 0.123 0.08 0.058 0.047 0.044 0.042 0.042 0.04 0.039
    Arith. Mean 0.659 0.367 0.208 0.127 0.086 0.062 0.05 0.044 0.042 0.041 0.041 0.039
    Arith. Std.Dev. 0.03 0.007 0.009 0.01 0.01 0.004 0.003 0.0 0.001 0.002 0.001 0.001
    Arith. Net Mean 0.62 0.328 0.168 0.087 0.047 0.022 0.011 0.005 0.003 0.001 0.002  
    Particles / OD                      
    Number of Particles 235294117.647 117647058.824 58823529.412 29411764.706 14705882.353 7352941.176 3676470.588 1838235.294 919117.647 459558.824 229779.412
    Mean particles / Abs600 379659729.967 358953650.11 349101064.758 337097589.752 314564328.405 326797385.621 350140056.022 386996904.025 334224598.93 367647058.824 131302521.008
    Mean of med-high levels:   337302803.729                  
    Table 3. The data of the Fluorescein standard curve experiment
    Fluorescein uM 10.0 5.0 2.5 1.25 0.625 0.312 0.156 0.078 0.039 0.02 0.01 0.0
    Replicate 1 77669.0 38692.0 20601.0 10396.0 5058.0 1898.0 1532.0 760.0 382.0 192.0 100.0 10.0
    Replicate 2 80872.0 37847.0 20419.0 10215.0 5014.0 2484.0 1212.0 618.0 329.0 155.0 81.0 11.0
    Replicate 3 80348.0 39774.0 19819.0 10018.0 4892.0 2221.0 1059.0 522.0 256.0 127.0 65.0 11.0
    Replicate 4 78408.0 39810.0 19893.0 10190.0 4987.0 2525.0 1201.0 608.0 309.0 159.0 97.0 12.0
    Arith. Mean 79324.25 39030.75 20183.0 10204.75 4987.75 2282.0 1251.0 627.0 319.0 158.25 85.75 11.0
    Arith. Std.Dev. 1530.028 944.399 386.012 154.677 70.22 289.269 199.872 98.583 52.083 26.625 16.153 0.816
    Arith. Net Mean 79313.25 39019.75 20172.0 10193.75 4976.75 2271.0 1240.0 616.0 308.0 147.25 74.75  
    Fluorescein/a.u.                      
    Fluorescein uM 10.0 5.0 2.5 1.25 0.625 0.312 0.156 0.078 0.039 0.02 0.01
    uM Fluorescein/a.u. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
    Mean uM fluorescein / a.u.:   0.0                  
    MEFL / a.u.:   768289204.589                  
    Table 4. The data of the plate reader measurement experiment
    Fluorescence Raw Readings:                  
    Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
    Colony 1, Replicate 1 148.0 293.0 262.0 323.0 149.0 621.0 315.0 199.0 148.0
    Colony 1, Replicate 2 149.0 293.0 261.0 318.0 146.0 615.0 307.0 195.0 146.0
    Colony 1, Replicate 3 150.0 283.0 258.0 315.0 150.0 611.0 305.0 224.0 145.0
    Colony 1, Replicate 4 152.0 290.0 254.0 316.0 149.0 619.0 335.0 223.0 140.0
    Colony 2, Replicate 1 153.0 296.0 271.0 298.0 154.0 594.0 310.0 206.0 153.0
    Colony 2, Replicate 2 155.0 293.0 259.0 310.0 151.0 580.0 301.0 199.0 147.0
    Colony 2, Replicate 3 155.0 290.0 258.0 315.0 148.0 589.0 298.0 208.0 146.0
    Colony 2, Replicate 4 154.0 290.0 258.0 310.0 154.0 615.0 313.0 202.0 145.0
                       
    Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
    Colony 1, Replicate 1 174.0 1097.0 766.0 1409.0 192.0 3976.0 794.0 591.0 143.0
    Colony 1, Replicate 2 187.0 1161.0 801.0 1486.0 204.0 4135.0 835.0 619.0 144.0
    Colony 1, Replicate 3 188.0 1170.0 799.0 1548.0 206.0 4242.0 830.0 624.0 149.0
    Colony 1, Replicate 4 195.0 1196.0 850.0 1504.0 201.0 4279.0 836.0 624.0 143.0
    Colony 2, Replicate 1 179.0 1115.0 1732.0 2000.0 190.0 4127.0 793.0 603.0 147.0
    Colony 2, Replicate 2 187.0 1152.0 1675.0 2071.0 207.0 4478.0 821.0 616.0 149.0
    Colony 2, Replicate 3 190.0 1105.0 1734.0 2090.0 203.0 4370.0 823.0 617.0 151.0
    Colony 2, Replicate 4 185.0 1157.0 1705.0 2105.0 204.0 4386.0 838.0 624.0 143.0
    Abs600 Raw Readings:                  
    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 0.072 0.068 0.059 0.071 0.067 0.06 0.056 0.063 0.045
    Colony 1, Replicate 2 0.07 0.069 0.055 0.07 0.067 0.062 0.059 0.067 0.044
    Colony 1, Replicate 3 0.071 0.068 0.057 0.07 0.066 0.059 0.056 0.074 0.047
    Colony 1, Replicate 4 0.071 0.071 0.057 0.07 0.068 0.06 0.059 0.075 0.048
    Colony 2, Replicate 1 0.069 0.068 0.057 0.066 0.067 0.062 0.056 0.065 0.047
    Colony 2, Replicate 2 0.07 0.069 0.057 0.067 0.066 0.061 0.056 0.063 0.05
    Colony 2, Replicate 3 0.072 0.068 0.055 0.065 0.064 0.061 0.057 0.065 0.043
    Colony 2, Replicate 4 0.071 0.071 0.058 0.068 0.064 0.065 0.057 0.063 0.045
                       
    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 0.329 0.288 0.126 0.291 0.301 0.251 0.239 0.267 0.045
    Colony 1, Replicate 2 0.332 0.293 0.125 0.298 0.313 0.254 0.25 0.28 0.044
    Colony 1, Replicate 3 0.331 0.292 0.125 0.302 0.31 0.256 0.244 0.276 0.047
    Colony 1, Replicate 4 0.339 0.296 0.13 0.3 0.309 0.257 0.246 0.278 0.047
    Colony 2, Replicate 1 0.308 0.294 0.184 0.313 0.294 0.25 0.248 0.264 0.046
    Colony 2, Replicate 2 0.32 0.298 0.186 0.321 0.305 0.266 0.259 0.267 0.045
    Colony 2, Replicate 3 0.321 0.289 0.182 0.318 0.299 0.259 0.258 0.268 0.043
    Colony 2, Replicate 4 0.314 0.301 0.186 0.323 0.304 0.262 0.26 0.27 0.045
    Table 5. The data of CFU experiment
      Pos. Control 1-1     Pos. Control 1-2     Pos. Control 1-3    
    Starting Sample's OD 0.096     0.064     0.11    
    Dilution order Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5
    Colony Count 273.0 24.0 4.0 130.0 9.0 2.0 161.0 21.0 1.0
    CFU*10^8/ml 0.218 0.192 0.32 0.104 0.072 0.16 0.129 0.168 0.08
      Pos. Control 2-1     Pos. Control 2-2     Pos. Control 2-3    
    Starting Sample's OD 0.096     0.106     0.099    
    Dilution order Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5
    Colony Count 174.0 15.0 4.0 150.0 21.0 1.0 133.0 22.0 5.0
    CFU*10^8/ml 0.139 0.12 0.32 0.12 0.168 0.08 0.106 0.176 0.4
      Neg. Control 1-1     Neg. Control 1-2     Neg. Control 1-3    
    Starting Sample's OD 0.089     0.121     0.103    
    Dilution order Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5
    Colony Count 102.0 7.0 2.0 151.0 28.0 1.0 241.0 46.0 2.0
    CFU*10^8/ml 0.082 0.056 0.16 0.121 0.224 0.08 0.193 0.368 0.16
      Neg. Control 2-1     Neg. Control 2-2     Neg. Control 2-3    
    Starting Sample's OD 0.11     0.103     0.106    
    Dilution order Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5 Dilution 3 Dilution 4 Dilution 5
    Colony Count 403.0 68.0 5.0 169.0 24.0 4.0 130.0 18.0 3.0
    CFU*10^8/ml 0.322 0.544 0.4 0.135 0.192 0.32 0.104 0.144 0.24

  • Analysis

    1. Abs600
    2. Fluorescence
    3. Comparison between different promoters and RBS
    4. CFU per OD600

  • Conclusion

    As the results show, each of test devices grows well, and can express reporter protein GFP normally (Figure 1, 2). Compared with different promoters (BBa_J23101, BBa_J23106 and BBa_J23117) and RBSs (BBa_B0034 and BBa_J364100). As for the promoters comparison, the strength of BBa_J23101 is the highest, and BBa_J23117 is the lowest. As for the RBSs comparison, the activity of BBa_J364100 is stronger than BBa_B0034. The activity of Device 4 is the highest, and Device 3 is the lowest.

    As the results show, each of test devices grows well, and can express reporter protein GFP normally (Figure 1, 2). Compared with different promoters (BBa_J23101, BBa_J23106 and BBa_J23117) and RBSs (BBa_B0034 and BBa_J364100). As for the promoters comparison, the strength of BBa_J23101 is the highest, and BBa_J23117 is the lowest. As for the RBSs comparison, the activity of BBa_J364100 is stronger than BBa_B0034. The activity of Device 4 is the highest, and Device 3 is the lowest.

    Besides, as for the CFU counts experiment, the colony counts increase with the elevated concentration of bacterium. However, theoretically speaking, the value of CFU/mL of each device should be equivalent, our result show that it is affected by error.

    We are looking forward to the results from the iGEM labs all over the world!