Team:TU Delft/Interlab

iGEM TU Delft

InterLab

The third International InterLab Measurement Study in Synthetic Biology.

Results

For this study, we measured three different test device and a negative- and positive control. We tranformed the five different plasmids into homemade TOP10 E.coli cells. The plasmids of the three test devices consist of a constitutive Anderson promoter with low (Bba_J23117), medium (Bba_J23106) or high (Bba_J23101) promoter strengths expressing GFP (Bba_I13504) as reporter in the pSB1C3 backbone. The positive control is a plasmid that consists of a promoter and GFP (Bba_I20270) in the pSB1C3 backbone, and the negative control a plasmid that consists of a TetR repressible promoter (Bba_R0040) in the pSB1C3 backbone. These plasmids were delivered to us with the iGEM distribution kit. From the transformation plates we picked eight colonies of every plate and did a colony PCR and gel electrophoresis. We then picked three colonies of each plate that had the insert, and let them grow overnight in liquid LB.

On the day of the measurements we measured the OD600 of the overnight cultures from two colonies of each device and diluted them according to the normalisation tables from the InterLab sheet.

After diluting we took 200µL culture out of every tube and put the tubes in the incubator for six hours and removed 200µL of culture every hour.

We then proceeded to measure the Abs600 of LUDOX and MiliQ and the fluorescence of 1x FITC with the plate reader according to the InterLab Plate Reader Protocol. See table 1 and 2. The fluorescence was measured with an excitation wavelength of 475 ± 9 nm and an emission wavelength of 509 ±20 nm, and a gain of 67.

Table 1: The Abs600 of LUDOX and MiliQ
LUDOX
Table 2: The fluorescence of FITC in PBS for different concentrations (µM)
FITC

With the LUDOX measurement we were able to convert the measured Abs600 to OD600 with a reference OD600. With the fluorescence of FITC we were able to create a standard curve to be able to convert the fluorescence of the cells into a concentration of GFP (see figure 1).

standard curve
Figure 1: The standard curve created with FITC to convert fluorescence into concentration

Hereafter we measured the Abs600 and fluorescence of all of the cell samples we took (see figure 2). We measured the fluorescence with the same settings as the FITC.

OD600
Figure 2: The fluorescence vs. OD600 of the cells for six hours of growing

We fitted the fluorescence of the cells to the standard curve to calculate the concentration of GFP in the cells (see figure 3 and table 3).

Standardcurve
Figure 3: The fluorescence of GFP fitted to the FITC standard curve
Table 3: The concentration of GFP in the cell cultures for six hours of growing
concentration

Discussion

The fluorescence per OD600 of the different devices increased with promoter strength as expected. Also as expected did the promoter strength affect cell growth: cells with a stronger promoter grew at a lower rate. This can be seen in figure 2, Test Device 1 grew around 5 times less but fluorescence was only 2 times smaller than Test Device 2. Thus, Test Device 1 has a promoter that is about 2.5 times stronger than Test Device 2. Test Device 2 grew about as fast as Test Device 3 but its fluorescence was around 200 times higher. This means that Test Device 1 has about 500 times as much fluorescence as Test Device 3, whose florescence was only slightly higher than that of the negative control.

Protocols used

For this study the protocols used were the InterLab Plate Reader Protocol and our own protocols for transformation and growing cells.

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