Team:Utrecht/InterLab

iGEM Team Utrecht 2018 participated in the Fifth International Interlab Measurement Study in synthetic biology for the Bronze Medal Requirements. Each year, the overall goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same laboratory. These measurements for example include fluorescence and absorbance, measurements which played a major role in this years’ interlab study.

When the fluorescence of a bacterium is measured by a plate reader, it is very important to take into account that this is an aggregate measurement of an entire population of cells. For this, the total fluorescence needs to be divided by the number of cells in order to determine the mean expression level of GFP per cell. This is usually determined by measuring the absorbance at 600nm, from which the optical density (OD) is calculated as an approximation of the number of cells. Since it is very unclear whether the OD is a good approximation of the number of cells, new measurements for the total number of cells needs to be tested. For this reason the research question for this year's InterLab study is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?


On August 16th all data were officially accepted by the iGEM Measurement Committee.

Materials

  • iGEM Measurement Kit
  • iGEM Parts Distribution Kit Plates
  • 1x PBS (phosphate buffered saline, pH 7.4 - 7.6)
  • ddH​2​O (ultrapure filtered or double distilled water)
  • Competent cells (​Escherichia coli​ strain DH5α)
  • LB (Luria Bertani) media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
  • 50 ml Falcon tube (covered in foil to block light)
  • Incubator at 37°C
  • 1.5 ml eppendorf tubes
  • Ice bucket with ice
  • Micropipettes
  • Micropipette tips
  • 96 well plates, black with clear flat bottom preferred

Methods

The fifth interlab study was performed according to iGEM protocols. No adjustments have been made:

  • Protocol to create our competent cells
  • Protocol for the transformation of competent E. coli
  • Protocol for the plate reader and CFU

Results

Table 1: OD600 reference point
Ludos CL-X
 H20
Replicate 1 0.093 0.068
Replicate 2 0.085 0.071
Replicate 3 0.091 0.070
Replicate 4 0.096 0.069
Arith. Mean 0.091 0.070
Corrected Abs600 0.022
Reference OD600 0.063
OD600/Abs600 2.900

Results of the plate reader measurements were integrated in the Excel template file provided by iGEM, additional CFU results were submitted via online form IV provided by iGEM.

Figure 1: Particle Standard Curve (linear scale)
Figure 2: Particle Standard Curve (log scale)
Figure 3: Fluorescein Standard Curve (linear scale)
Figure 4: Fluorescein Standard Curve (log-scale)
Table 2: Fluorescence Raw Readings at t=0H
t = 0 -
 +
 D1 D2 D3
 D4 D5 D6 Blank
C1R1 680 785 969 831 802 1048 1000 1000 686
C1R2 675 743 908 821 674 1014 1006 1006 686
C1R3 682 762 898 806 655 1014 993 993 691
C1R4 675 769 921 815 687 1018 1015 1015 710
C2R1 685 755 1007 885 695 1066 794 794 706
C2R2 650 746 958 796 673 984 760 760 697
C2R3 674 750 942 779 692 1034 771 771 699
C2R4 690 747 996 808 683 1040 779 779 694
Table 3: Fluorescence Raw Readings at t=6H
t = 6
 -
 +
 D1 D2 D3
 D4 D5 D6 Blank
C1R1 982 5454 2006 5451 958 2134 2235 2610 697
C1R2 928 5874 1891 5209 925 2070 2095 2791 654
C1R3 884 5612 1867 5309 915 2022 2097 2668 682
C1R4 878 5145 1873 5277 928 2051 2168 2729 677
C2R1 957 4540 1732 5119 928 2325 1701 2800 689
C2R2 917 4391 1525 4971 911 2204 1728 2706 684
C2R3 885 4431 1655 5017 916 2202 1708 2671 682
C2R4 827 4020 1631 4688 949 2200 1696 2869 677
Table 4: Abs600 Raw Readings at t=0H
t = 0
 -
 +
 D1 D2 D3
 D4 D5 D6 Blank
C1R1 0.0774 0.0720 0.0723 0.0712 0.0741 0.0697 0.0729 0.0768 0.0742
C1R2 0.0790 0.0745 0.0760 0.0763 0.0764 0.0760 0.0792 0.0775 0.0745
C1R3 0.0822 0.0805 0.0761 0.0755 0.0770 0.0751 0.0801 0.0801 0.0768
C1R4 0.0776 0.0755 0.0726 0.0734 0.0728 0.0758 0.0737 0.0767 0.0686
C2R1 0.0787 0.0773 0.0784 0.0814 0.0777 0.0789 0.0793 0.0811 0.0744
C2R2 0.0777 0.0771 0.0774 0.0808 0.0770 0.0780 0.0769 0.0792 0.0726
C2R3 0.0836 0.0809 0.0729 0.0755 0.0718 0.0739 0.0723 0.0796 0.0691
C2R4 0.0829 0.0781 0.0788 0.0780 0.0749 0.0756 0.0754 0.0752 0.0724
Table 5: Abs600 Raw Readings at t=6H
t = 0
 -
 +
 D1 D2 D3
 D4 D5 D6 Blank
C1R1 0.480 0.380 0.240 0.430 0.460 0.150 0.250 0.370 0.067
C1R2 0.440 0.440 0.220 0.400 0.440 0.140 0.240 0.430 0.074
C1R3 0.420 0.400 0.230 0.410 0.430 0.140 0.230 0.400 0.073
C1R4 0.420 0.350 0.210 0.400 0.440 0.140 0.240 0.410 0.065
C2R1 0.510 0.440 0.200 0.440 0.420 0.160 0.220 0.410 0.073
C2R2 0.470 0.420 0.170 0.410 0.410 0.150 0.220 0.380 0.070
C2R3 0.450 0.420 0.190 0.410 0.420 0.150 0.210 0.390 0.069
C2R4 0.390 0.380 0.190 0.400 0.420 0.150 0.220 0.410 0.068

Colony Forming Units

As expected, all triplicates of both negative controls and positive controls showed colonies in 10x dilution series. Dilutions with the following Final Dilution Factor were used: 8x10^4 (d3), 8x10^5 (d4), 8x10^6 (d5). CFU values for all triplicates of negative and positive controls are shown in table 6. In Figure 5abc an example of the colony growth as seen from the LB + Cam plates is shown.

Figure 5: Negative Control (BBa_R0040) cultures (3.2) colony growth on LB+Cam plates in 10x dilution series: 8x10^4 (d3), 8x10^5 (d4), 8x10^6 (d5). Colony count: d3-201, d4-73, d5-11
Table 1: OD600 reference point
Name Dilution Factor #Colonies Name Dilution Factor #Colonies
1.1 8x10^4 >300 3.1 8x10^4 >300
1.1 8x10^5 276 3.1 8x10^5 159
1.1 8x10^6 33 3.1 8x10^6 8
1.2 8x10^4 >300 3.2 8x10^4 201
1.2 8x10^5 179 3.2 8x10^5 73
1.2 8x10^6 22 3.2 8x10^6 11
1.3 8x10^4 >300 3.3 8x10^4 >300
1.3 8x10^5 226 3.3 8x10^5 95
1.3 8x10^6 36 3.3 8x10^6 16
2.1 8x10^4 >300 4.1 8x10^4 >300
2.1 8x10^5 300 4.1 8x10^5 54
2.1 8x10^6 30 4.1 8x10^6 11
2.2 8x10^4 >300 4.2 8x10^4 >300
2.2 8x10^5 114 4.2 8x10^5 82
2.2 8x10^6 22 4.2 8x10^6 14
2.3 8x10^4 >300 4.3 8x10^4 >300
2.3 8x10^5 154 4.3 8x10^5 191
2.3 8x10^6 40 4.3 8x10^6 13
Results
Lab Safety