Template:2018_NAU-CHINA

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InterLab

Introduction

Reliable and repeatable measurement is a key component to all engineering disciplines. Based on this idea, the measurement committee encourages all competing teams to participate in the InterLab study. This year, the committee wants iGEMers from around the world to answer the following question together: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

Materials

1.Measurement Kit (provided with the iGEM distribution shipment) containing:
1ml LUDOX CL-X
150 μL Silica Bead (microsphere suspension)
Fluorescein (powder, in amber tube)
2. iGEM Parts Distribution Kit Plates
3. 1x PBS (phosphate buffered saline, pH 7.4 – 7.6)
4. ddh1O (ultrapure filtered or double distilled water)
5. Competent cells (Escherichia coli strain DH5α)
6. LB (Luria Bertani) media
7. Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
8. 50 ml Falcon tube (covered in foil to block light)
9. Incubator at 37°C
10. Plate reader (SpectraMax i3x)
11. 1.5 ml eppendorf tubes
12. Ice bucket with ice
Micropipettes (capable of pipetting a range of volumes between 1 μL and 1000 μL)
Micropipette tips
96 well plates, black with clear flat bottom

Calibration

1. OD600 Reference point

We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform the absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer.

Table 1. The data for OD600 measured by our plate reader, with a ratiometric conversion factor of 3.505.

2. Particle Standard Curve

We prepared a dilution series of monodisperse silica microspheres as instructed in the protocol.

Figure 1. Serial dilution of monodisperse silica microspheres.

The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.

Table 2. The data for Abs600 measured by our plate reader.

Figure 2-1. Abs600 is plotted against the particle count.

Figure 2-2. Abs600 is plotted against the particle count on a logarithmic scale.

3. Fluorescence standard curve

We prepared a dilution series of fluorescein as instructed in the protocol.

Figure 3. Serial dilution of monodisperse silica fluorescein.

By measuring the serial dilutions in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We were able to use this to convert our cell based readings to an equivalent fluorescein concentration.

Table 4. The data for fluorescence measured by our plate reader.

Figure 4-1. Fluorescence is plotted against the fluorescein concentration.

Figure 4-2. Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.

Cell Measurement

Transformation

We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5α cells. DH5α competent cells were bought from Transgen Biotech. The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates.

Cell Culture

We picked 2 colonies from each of the transformation plates and inoculate in 5 mL LB medium + Chloramphenicol. The cells were grown overnight 16 hours at 37°C and 220 rpm.

Sampling

We made a 1:10 dilution of each overnight culture in LB + Chloramphenicol and measured Abs600 of these 1:10 diluted cultures. After measuring, we further diluted these cultures to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tubes. These falcon tubes were covered in foil to block light. We took 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes prior to incubation. These samples were put on ice before measurement was taken. Then we incubated the remainder of the cultures at 37°C and 220 rpm for 6 hours. After these procedures were finished, we took 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes and placed them on ice. At the end, we carried out our measurement. Samples were laid out in 96 well plates according to the diagram below:

Figure 5. Samples were laid out in 96 well plates as shown.

Table 7-1. Fluorescence raw readings at hour 0.

Table 7-2. Abs600 raw readings at hour 0.

Table 7-3. Fluorescence raw readings at hour 6.

Table 7-4. Abs600 raw readings at hour 6.

After some calculation, we got some experimental values on uM Fluorescein / OD:

Table 8-1. uM Fluorescein / OD at hour 0.

Table 8-2. uM Fluorescein / OD at hour 6.

CFU

In this part, we want to know whether OD600 can be calibrated to colony forming unit (CFU) counts. We counted two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.

We first diluted our overnight culture to the linear detection range of our plate reader by adding 25 μL culture to 175 μL LB + Cam in a well in a black 96-well plate. Then we further diluted them to OD600 = 0.1 in 1mL of LB + Cam media and did this in triplicate for each culture.

Figure 6. Check whether the OD600 is 0.1 (minus the blank measurement).

After ensuring all the OD600 is 0.1, we performed serial dilutions and incubation for them as directed.

Figure 7. Serial dilutions and plates for these samples.

Here are our data:

•Discussion

We did our experiments twice. The first attempt was not so successful as the bacteria grew faster than we assumed. We were not able to keep them at a target OD. Our laboratory is on the first floor, while the plate reader is on the sixth floor. During this period, when we read the number from the machine and ran upstairs to do dilution, the OD had already changed a lot. So we recommend using an ice pack to slow down cell growth in order to obtain better data. All the dilutions and transporting may be performed on ice, just as we did in our second attempt. Additionally, we were not so optimistic about the results of serial dilution and CFU counting as the dilution may not be so accurate, and are easy to be affected by complicated reasons. Our data still weren’t look so perfect although we made every step quite carefully and did a second try.

Team:NAU-CHINA/InterLab