Team:TecMonterrey GDL/InterLab

InterLab

This year (2018) the iGEM Team TecMonterrey_GDL decided to contribute into the Interlab study measurements. The goal of the study is summarized in achieving a way to reduce lab-to-lab variability in fluorescence measurements via normalizing to absolute cell count or colony forming units. The approaches taken were indicated by the iGEM measurement team: 1. Converting between absorbance of cells to absorbance of a known concentration of microsphere silica beads 2. Counting colony-forming units (CFU) from the samples The team followed a series of protocols created to help standardize the measurement of GFP (green fluorescent protein), which is widely used as measurement marker. Specifications: The equipment we used was Cytation 3 by Biotek. It can measure both absorbance and fluorescence. And the pathlength correction was disabled. The temperature of the measurements was done at room temperature.

Calibration Protocol 1: OD600 Reference point - LUDOX Protocol

The first protocol’s goal was to obtain a conversion factor between absorbance measured and the OD600 measurement as it occurs in spectrophotometry measurements. This was made with the kit provided LUDOX CL-X (45% colloidal silica suspension) provided by iGEM and sterile mili Q water, reactants were added accordingly to the protocol and the obtained results are shown in the next table (Table 1):

Table 1. Calibration Protocol 1: OD(600) Reference point - LUDOX Protocol

The corrected absorbance was was calculated by subtracting the mili Q water reading. The reference OD600 was provided to the team by iGEM. The correction factor was obtained by dividing the reference OD600 by corrected Abs600 obtained.

Calibration Protocol 2: Particle Standard Curve - Microsphere Protocol

Silica microspheres were provided by iGEM kits, these were meant to simulate cells in a dilution, and thus the protocol stated to make serial dilutions of the microsphere suspension in order to construct a standard curve for Abs600 which would allow conversion of Abs600 measurements into an estimated number of cells. Our curve results are shown in the following figure:

Figure 1. Calibration Protocol 2: Particle standard curve

Microsphere particle standar curve If you wish to see the Excel sheet you can download it here: Excel

Calibration Protocol 3: Fluorescence Standard Curve - Fluorescein Protocol

Due to the expenses and stability of the GFP, fluorescein, a small molecule with similar properties to GFP, was provided in the iGEM kit for teams to make the indicated serial dilutions standard curves. The preparation of the stock solution with PBS and measurements were made according to protocols, the resulting curve is meant to serve as a way to convert cell based readings to equivalent fluorescein concentration. The results are shown in Figure 2.

Figure 2. Calibration Protocol 3: Fluorescence Standard Curve

Fluorescence curves If you wish to see the Excel sheet you can download it here: Excel

Cell measurement protocol

For this protocol, transformations with the required devices (6 + positive and negative control) in Distribution kits were made. For transformations, the iGEM team followed recommended iGEM protocols. After picking two colonies of each device, they were inoculated in 10 ml of LB medium + chloramphenicol and grown overnight at 37°C and 220 rpm. Then cells from all cultures were diluted to 1:10, absorbance was measured and the team proceeded to dilutions until 0.02 Abs was obtained. Samples were gathered from these last cultures as “Time 0” and then incubation for 6 hours proceeded. After 6 hours, samples were gathered and measurements were made following the instructions and layouts provided in the protocol. The results from this experiment are shown in Figure 3.

Cell measurement Protocol

If you wish to see the Excel sheet you can download it here: Excel

Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

This protocols were made to calibrate OD600 to colony forming units, which are related to culture concentrations, the protocol was made under the assumption that 1 bacterial cell gives rise to one colony. The team took samples from the overnight cultures of the positive and negative controls, measured absorbance and diluted to obtain OD600 of 0.1 in 1ml of LB+ chloramphenicol media with the help of CV = CV relationship and did this by triplicate and obtained measurements. For the CFU, from 3 mL overnight cultures of the positive and negative controls, “starting samples” were made by diluting the cultures until OD600 = 0.1, then indicated serial dilutions of consequential 1:10 dilutions were made until dilution 5 was reached (8 x 10^-5 factor). The dilutions from 3 to 5 ( 8 x 10^-3, 8 x 10^-4 and 8 x 10^-5 factors, respectively) were cultured aseptically for 20 hours in LB + chloramphenicol agar plates. A total of 36 plates was obtained in total. CFU/mL calculations followed, after counting colonies, the colony number was multiplied by the final dilution factor and results were delivered.