Difference between revisions of "Team:Uppsala/Interlab"

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Introduction

We are happy to say that we now have re-introduced the Uppsala team to the interlab study after a year in absence. The flow cytometer that has been used by the Uppsala iGEM team earlier years was unavailable this year. However, we had access to a fluorescence plate reader which allowed us to participate in the interlab study using the plate reader protocol.

Method

Thorough instructions for the interlab study are available at iGEM.org. The form for providing the results from the interlab study is available here.

The protocol consists of two calibration protocols: OD600 reference point and FITC fluorescence standard curve, and a cell measurement protocol. In each protocol, there are instructions on how to perform measurements with a plate reader or a cuvette reader. Five different devices were provided by iGEM and included in the distribution kit. Positive control J23151.B0032.E0040.B0010.B0012 Negative control R0040 Device 1 J23101.B0034.E0040.B0015 Device 2 J23106.B0034.E0040.B0015 Device 3 J23117.B0034.E0040.B0015 In addition to this FITC standard and LUDOX solution was provided in the kit and used for calibration. A Shimadzu UV-1800 cuvette spectrophotometer was used for calibration and measurement of OD600. A Fluoroscan Ascent was used for the calibration and measurement of fluorescence.

OD600 Reference point

Was performed as instructed in cuvettes.

FITC fluorescence standard curve

While preparing the FITC solution, the solution was incubated overnight to properly dissolve the FITC into 1X PBS. This was recommended in a note in the official protocol if the FITC was not dissolved after 4 hours. The rest of the protocol was performed as instructed in the Fluoroscan Ascent plate reader.

Settings used: Excitation wavelength: 485 nm Emittation wavelength: 538 nm Scaling factor: 0.7/1 Integration time was varied between 20 ms and 80 ms. The variation in integration time did not cause a big difference on the results. 40 ms integration time was chosen for the presented results.

Cell measurement protocol

The first day, E.coli DH5α was transformed with the five different devices provided by iGEM with our transformation protocol for chemically competent E.coli DH5α. The next day, 2 colonies of each device was inoculated into 5 ml LB+Chloramphenicol in a 50 ml Falcon tube. The lid of the tube was screwed down loosely and the tube was placed on a shaking table at 220 rpm overnight.

The third day, OD600 of each culture was measured in the calibrated cuvette spectrophotometer. Calculations were made with the provided excel sheet and each tube was diluted with LB+Cloramphenicol to 10 ml of OD600 0.02 as instructed in 50 ml Falcon tubes. 100 μl samples were then collected each hour and kept on ice in 4℃ for later measurement. When 7 samples had been collected, measurements were done in the calibrated Fluoroscan Ascent plate reader. Both cultures of replicates with device 3 was notably clearer than all other cultures after 6 hours.

No measurements of OD600 were done on the plates. This was due to a misunderstanding of the protocol, and not enough sample volume was available to be able to do OD600 measurements in the cuvette spectrophotometer.

Results

Since no OD600 measurement was done on the hourly samples, we have no Fl/Abs600 data. However, we can still compare the arbitrary fluorescence units of the different measurements to each other.

Figure 2. The mean fluorescence of the two replicates for each device (n=2), corrected for the readout of LB media with 25 μg/ml chloramphenicol. Error bars show standard deviation.

Discussion

In figure 1 and figure 2, we can see that both replicates of device 3 have a fluorescence close to the values of the negative control. This is probably due to low growth of cells, since the culture looked clear even after 6 hours of incubation. Why this happened in both replicates is unknown. While providing a mean and standard deviation of only two replicates is not very good form, figure 2 is a lot easier to read than figure 1. In figure 2, it is clear that device 2 has a higher fluorescence than device 1. Since only 100 μl was sampled (as instructed in protocol) from each replicate every hour and the plate reader assay is volume dependent, a better consistency in volume could have been achieved by sampling a larger volume. This would allow for some leftovers in the sample tube, instead of assuming that it is possible to extract the total volume from a sample of 100 μl every time. We decided to keep the samples in eppendorf tubes that were spun down before pipetting down into the plate for measurements, but sampling more than 100 μl would have solved this issue completely. The interlab study in the Uppsala team was performed by Fredrik Lindeberg.