Difference between revisions of "Team:SIAT-SCIE/HP/Silver"

The interlab study for iGEM 2017 is to analysis the relationship of fluorescein of device 1-6 over time with a similar initial OD600 of 0.02.

The Construction of OD600 Reference Point

-Protocol:

1. Add 100ul of LUDOX into wells A1, B1, C1, D1
2. Add 100ul of H2O into wells A2, B2, C2, D2
3. Use plate reader to measure the absorbance 600 nm

-Results:

Plate Reader

Build Up Fluorescein Fluorescence Standard Curve

-Introduction:

By carrying out a dilution of the fluorescein with known concentration, we are able to plot a fluorescence standard curve which can be used to analyze the fluorescence level of the GFP.

-Protocol:

1. Prepare 2X of fluorescein stock solution
2. Prepare 1X solution by mixing 1ml of 2X solution with 1ml of 1XPBS
3. Add 200ul of 1X fluorescein solution in wells A1, B1, C1, D1
4. Add 100ul of PBS in wells A2-A12
5. Transfer 100ul of fluorescein solution into well A2 and mix gently
6. Repeat until well A11 and then transfer 100ul of the solution from A11 to waste
7. Repeat with rows B, C, D
8. Measure fluorescence (excitation: 488 emission: 507) with a plate reader

-Results:

We weren’t managed to measure the fluorescein of the first two columns because the values exceeded the maximum value of the plate reader, shown as ‘OVERFLOW’. This error may due to incorrect settings of the GFP wavelength. We tried settings such as 488/509 and 488/507 but none of them allow us to measure the value for the first two column.

Cell measurement

-Introduction:

We have 2 groups of cultures of device 1-6, positive control and negative control. By incubating them from similar OD600s, we took samples from different time points and tried to investigate the change of OD600 and fluorescence over time.

-Protocol:

1. Transform E.Coli DH5a with the required 8 plasmids
2. Pick 2 colonies from each plate and inoculate on 3ml LB+CHL
3. Grow the cell overnight at 37 degree Celsius 220 rpm
4. Measure the OD600
5. Carry out dilution to a target OD600 of 0.02 with a total volume of 3ml for each cultures
6. Incubate at 37°C and 220 rpm
7. Take samples of 500ul from each cultures at hour 0h, 2h, 4h and 6h
8. Place all the samples on an ice box
9. Add samples to a 96-well plate to measure OD600 and fluorescence of each time point

-Results:

The OD600 increases over time, but the fluorescence decreases over time.

-Problems:

As shown on the fluorescence-background table, we got results with negative values because the fluorescence of the cultures were all lower than that of the LB. As a result, we weren’t able to analysis the change of fluorescence over time since we believed that the GFP wasn’t expressed. We repeated the cell measurement experiment for two times and they both gave similar results with negative values of the background fluorescence. We believed that those errors could due to the incorrect settings of our plate reader as mention in part two. Besides, the 2017 interlab protocol required us to block light when incubating the culture. However, we might be failed to do that because the cultures were exposed to light when we were sampling and during the incubation.

Another problem is about device 1. The plasmid was provided in the plate 7 21F of the kit. However, during our experiment, we failed to have device 1 grown during the transformation. We carried out the transformation for 3 times and only got a few colonies from one of them. We believed that the failure of the transformation of device 1 was because we didn’t add enough plasmid into the DH5a.

To Download the full version of interlab data, click here.