Difference between revisions of "Team:Manchester/InterLab"

Fluorescence assays are amongst the most important measurement tools in biological experiments. However, the direct comparison of fluorescence data is problematic as data is often reported in different units or processed in different ways. This inability to directly compare results makes it harder to work collaboratively between laboratories and thus hinders the advancement of research. Therefore, it is important that a standardised protocol be established, so that groups around the world can achieve better, more streamlined fluorescence measurements. The Fourth Interlaboratory Measurement study aims to test the effectiveness of this standardised protocol by answering the important question: How close can the numbers be when fluorescence is measured all around the world using the same exact protocol?

Manchester iGEM 2017 is proud to have taken part in the interlab study for the first time. We hope our participation will help benefit other iGEM teams and the synthetic biology community as a whole. Back to top

Protocols

Aseptic technique was maintained at all times.

Transformation

The E.coli strain used was Dh5α. The DNA provided in the distribution kit for the transformations was re-suspended using 10μl of dH2O. Competent cells were transformed using the following 8 devices, inside the plasmid backbone pSB1C3:

- BBa_R0040 (Negative Control)

- BBa_I20270 (Positive Control)

- BBa_J364000 (Device 1)

- BBa_J364001 (Device 2)

- BBa_J364002 (Device 3)

- BBa_J364003 (Device 4)

- BBa_J364004 (Device 5)

- BBa_J364005 (Device 6)

Transformations were done using our refined protocol (see additional work) and the transformed cells were subsequently plated on 8 LB agar plates containing chloramphenicol at a concentration of 25mg/ml. The plates were incubated overnight at 37°C.

Overnight Cultures

5ml of sterile LB broth was transferred to a total of 16 x 50ml falcon tubes, chloramphenicol was then added to each tube at a concentration of 25mg/ml. Two colonies were picked from each plate, each individual colony was transferred to a separate falcon tube. The tubes were incubated overnight at 37°C and 180 rpm.

Plate Reader Settings

Measurements of OD600 and fluorescence were taken using a BMG LabTech Clariostar plate reader, provided to us by the Manchester Institute of Biotechnology (MIB). OD600 measurements were all taken at 24°C with 35 flashes, and an orbital averaging of 3. Fluorescence measurements were taken with 8 flashes and a gain of 748. Excitation and emission wavelengths were 515-20 nm and 470-15 nm respectively. Pathlength correction was turned off for both measurements.

OD600 Reference Point

LUDOX S-40 was used as a single reference point to calibrate the plate reader. A ratiometric conversion factor was obtained to ensure that all Abs600 measurements were converted to OD600 measurements, whilst also taking into account differences in instruments.

100 µl of LUDOX was transferred into wells A1-D1 of a black, clear-bottom 96-well plate. 100 µl of distilled H₂O was then added into wells A2-D2. Absorbance of LUDOX and H₂0 were then measured at 600nm. A ratiometric conversion factor of roughly 3.269 was obtained.

Fluorescence Standard Curve

The fluorescein stock was spun down for 30 seconds at 3000rpm and then re-suspended in 1mL of 1xPBS to produce a 2xstock solution (100μM). This was then further diluted with 1xPBS to a 1x stock solution with a final concentration of 50μM.

200µl of the fluorescein stock was transferred into wells A1-D1. The fluorescein stock was then serially diluted in four replicates (A2-A12, B2-B12, C2-C12, D2-D12) by mixing with 1x PBS to obtain 100µl of 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.048828125, and 0 µM of fluorescein solution. Fluorescence was then measured using a BMG LabTech ClarioStar plate reader, and a fluorescence standard curve generated (Figure 1). This standard curve was used to correct cell based readings to an equivalent fluorescein concentration and measure the concentration of GFP.

Cell Measurements

Overnight cultures of each device were diluted to a target OD of 0.02 and incubated at 37°C and 220 rpm, falcon tubes were wrapped in tin foil throughout. 500 µL of each device was transferred to an Eppendorf tube and put on ice at time points 0h, 2h, 4h, and 6h. 100μl of each sample was then transferred to 96 well plate using the provided set-up guidelines and measurements. OD and fluorescence measurements were taken simultaneously. Back to top

Results

Figure 1: Standard curve of fluorescein fluorescence generated via 10-fold serial dilution using 4 standard replicates.

Figure 2: Average OD corrected fluorescence measurements of colony 1 and 2. Error bars show the standard deviation.

Overall, the results of the experiment was rather unexpected. Out of all the devices tested, only device 2 exhibited the expected trend, the fluorescence of cells increasing with time. Device 4 and the positive control also display this trend up to 4 hours before decreasing. However, this decrease could be explained by our cells dying after 6 hours. The negative control displays fluorescence, which was not supposed to happen. We suspect that this may be due to contamination or autofluorescence of colony 2. Colony 1, which was not contaminated, showed very low levels of fluorescence, similar to those seen in device 3 and 6 (See Full Results).

Something worth noting is the large error seen at 4h in device 1. There is a massive spike in fluorescence despite the low OD detected in both colonies. In fact, based on our results, it could be observed that the number of cells is decreasing with time. We were also unable to find any non-arbitrary characterization data in the registry on part J23101 which is the promoter in device 1. However, we do know that it is a constitutive promoter and the strongest out of the three used for the study (J23117 in devices 3 and 6 , J23106 in devices 2 and 5), as seen from RFP fluorescence measurements at: http://parts.igem.org/wiki/index.php?title=Part:BBa_J23101.

We suspect that level of GFP expression was too high in our cells, causing a number of them to die but not before the protein could accumulate. This could also explain why device 4, which utilizes the same promoter but a with a bicistronic design element for more precise expression, exhibited a more ‘normal’ trend. However, we do not have sufficient evidence to substantiate this claim.

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Improvements

We are aware that contamination has occurred in colony 2 (Figure 3), as fluorescence was detected in the negative control (which does not have a GFP construct). This is most likely due to human error when transferring samples.

Additional work

We wanted to take full advantage of the interlab project and use it to improve upon the skills that we have gained since beginning the experimental part of our project. For this reason, we decided to make our own Dh5α competent cells. This also gave the rest of the team plenty of stock to work with whilst they continued to complete their own experiments. We used the Benchling protocol which can be found here: https://benchling.com/protocols/PqDdikG7/tss-competent-e-coli-preparation

Transformation

Initially we struggled to obtain colonies from the transformation of test kit 7, refining our protocol led us to successfully transform kit 6. This protocol has since been adopted by other members of our team to improve the efficiency of their own transformations. Our protocol is as follows:

- 2μl of DNA was added to 100μl of competent Dh5α E coli cells.

- Cells were incubated on ice for 30 minutes.

- Cells were heat shocked in water bath set at 42°C for 30 seconds.

- 800μl of SOC media was added and cells were incubated for 60 minutes at 37°C.

- Cells were then spun down in a tabletop centrifuge for 2 minutes at 5000 rpm.

- 600μl of the supernatant was removed and the pellet re-suspended in the remaining supernatant.

- The remaining 200μl of re-suspended cells were then plated.

Miniprep and restriction digest

As we initially struggled to transform all the devices in kit plate 7, transformations which yielded colonies were miniprepped and the purity and concentration of the DNA was assessed using a nanodrop spectrophotometer. We then performed a restriction digest (using XbaI and PstI) and ran the DNA on an agarose gel. This confirmed that our transformations had not been successful in all cases, as we predicted our expected band sizes using Snapgene and they did not match our results. Conversely, the successful transformation of all devices from kit plate 6 was confirmed using the same technique. At this point we then prepared overnight cultures ready for the plate reader measurement interlab protocol. Back to top

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   We suspect that level of GFP expression was too high in our cells, causing a number of them to die but not before the protein could accumulate. This could also explain why device 4, which utilizes the same promoter but a with a bicistronic design element for more precise expression, exhibited a more ‘normal’ trend. However, we do not have sufficient evidence to substantiate this claim.   </p>
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 <a href="https://static.igem.org/mediawiki/2017/0/0e/T--Manchester--Manchester_Interlab_2017_Measurements.xlsx" target="_blank"><button>DOWNLOAD FULL RESULTS</button></a>