InterLab

This year, the Cardiff iGEM team decided to participate in the InterLab study. This page shows our team's data.

Calibration 1: OD600 Reference point LUDOX Protocol

We carried out the LUDOX protocol as follows:

100 μl of LUDOX was pipetted into wells A1, B1, C1, D1
100 μl of ddH2O was pipetted into wells A2, B2, C2, D2
The absorbance was measured at 600 nm of all samples in the measurement mode using a BMG LABTECH CLARIOstart microplate reader
The data was recorded in a notebook and then imported into the Excel sheet provided ( OD600 reference point tab )

We obtained the following table from our data:

Figure 1. Results from the LUDOX protocol

Calibration 2: Particle Standard Curve – Microsphere Protocol

We carried out the Particle Standard Curve protocol as follows:

The silicia beads (300 μL) were vortexed for 30 seconds
96 μL microspheres were immediately pipetted into a 1.5 mL eppendorf tube
904 μL of ddH2O was added via pipette to the microspheres
The solution was further vortexed to make up the microsphere stock solution
100 μl of ddH2O was pipetted into wells A2, B2, C2, D2…. A12, B12, C12, D12
The tube containing the stock solution of microspheres was vortexed vigorously for 10 seconds
200 μl of microspheres stock solution was immediately pipetted into A1
100 μl of microsphere stock solution was transferred from A1 into A2
A2 was mixed by pipetting up and down 3x and 100 μl transferred into A3
The previous step was repeated for subsequent wells A3-A11
Finally after pipetting in well A11, 100 μl was transferred into liquid waste
The dilution series was repeated for row B,C,D
All wells were remixed before being placed into the plate reader
Abs600 of all samples was measured
The data was recorded in a notebook and imported into the excel sheet provided (Particle standard curve tab)
Data obtained at a gain of 150 at 37°C

We obtained the following table and curve from our data:

Figure 2. The particle standard curve and tables.

Calibration 3: Fluorescein standard curve – Fluorescein Protocol

We carried out the Fluorescein Standard Curve protocol as follows:

The fluorescein kit tube was spun to make sure pellet is at the bottom of tube
A 10x fluorescein stock solution (100 μM) was prepared by re-suspending fluorescein in 1 mL of 1xPBS
The 10x fluorescein stock solution was diluted with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS
100 μl of PBS was pipetted into wells A2, B2, C2, D2…. A12, B12, C12, D12
200 μl of fluorescein 1x stock solution was pipetted into A1, B1, C1, D1
100 μl of fluorescein stock solution was transferred from A1 into A2
A2 was mixed by pipetting up and down 3x and 100 μl transferred into A3
The previous step was repeated for subsequent well A3-A11
Finally after pipetting in well A11, 100 μl was transferred into liquid waste
The dilution series was repeated for row B,C,D
The fluorescence of all samples in instrument were measured
The data was recorded and imported into the excel sheet provided
Data obtained at a gain of 150 at 37°C

We obtained the following table and curve from our data:

Figure 3. The Fluorescein standard curve and tables.

Cell Measurements

We carried out the Cell Measurements protocol as follows:
On day 1 we transformed E. coli strain DH5 alpha with the InterLab plasmids. These are Kit Plate 7, wells 2D, 2B, 2F, 2H, 2J, 2L, 2N, and 2P.
On day 2, 2 colonies were picked from each of the transformation plates and inoculated in 10 ml of LB medium and chloramphenicol. The cells were grown overnight at 37 °C and 220 rpm.
On day 3, we performed the Cell growth, sampling, and assay. This is as follows:

A 1:10 dilution of each overnight culture in LB + Chloramphenicol was made
The ABS600 was measured of the 1:10 diluted cultures
The data was then recorded in a notebook
The cultures were then further diluted to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tubes
500 μL samples of the diluted cultures at 0 hours were taken into 1.5 ml eppendorf tubes, prior to incubation. The samples were then placed on ice
The remainder of the cultures were incubated at 37 °C and 220 rpm for 6 hours
500 μL samples of the cultures at 6 hours of incubation were taken into 1.5 ml eppendorf tubes and placed on ice
At the end of the smapling point the samples were measured for Abs600 and fluorescence
The data was recorded and imported into the excel sheet provided (fluorescence measurement tab)

These results produced the following tables. Note, we think that one of the colonies from Device 5 is an anomaly:

Figure 4. Our raw-value measurement tables for the InterLab study

Colony Forming Units per 0.1 OD600 E. coli cultures

Finally, we carried out the CFU protocol as directed, using only two positive control colonies (BBa_120270), and two negative controls (BBa_R0040).

A 1:8 dilution was made for each overnight culture in LB + Cam before measuring the OD600
25 μL of culture was added to 175 μL LB +Cam in a black 96- Well plate with a clear, flat bottom and a measurement taken in the plate reader at 600nm
The overnight culture was then diluted to OD600 = 0.1 in 1 mL of LB + CAM media. This was done in triplicate for each culture
This was accomplished by using (C1)(V1) = (C2)(V2) to calculate the dilutions, where: C1 is your starting OD600, C2 is your target OD600 of 0.1, V1 is the unknown volume in μL, and V2 is the final volume of 1000 μL
The amount of culture required to obtain OD600 = 0.1 was calculated for each culture
The OD600 was then measured in the following layout where the OD600 obtained was as expected, 0.1

Figure 5. The layout used for pipetting the plates.

Serial dilutions for the triplicate starting samples prior were made
This was done via 12 starting samples 6 positive and 6 negative controls
The dilutions were carried out as shown in the diagram below
100 μL of dilutions 3,4 and 5 were then aseptically spread on LB + Cam plates and incubated overnight at 37 °C

Figure 6. The diagram followed for the serial dilutions.

Finally the colonies on each plate with fewer than 300 colonies were counted after 20 hours of growth and the colony count multiplied by the final Dilution Factor on each plate to determine the CFU/ml/OD

From this, the following tables were generated:

Team:Cardiff Wales/InterLab

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