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Revision as of 23:24, 7 October 2018
Aim of the InterLab Study
Synthetic biology is an emerging and constantly developing field which holds great promise to solve major problems humanity is facing today in terms of biology related medicine. In order to make use of this potential it is very important to reduce measurement variability and ensure there are standards in the field. To contribute towards this, our team has taken part in the iGEM interlab study, which has the noble goal of identifying and correcting the sources of systematic variability in synthetic biology measurement.
iGEM has in previous interlab studies showed that it is possible to reduce the variability in measurements between labs by measuring GFP expression in absolute fluorescence units which are calibrated against a known concentration of a chromoprotein. However, there is still a large source of variability in such measurements depending on the number of cells in the sample. By dividing the total fluorescence by the number of cells we can determine the mean expression level of GFP per cell. Usually, computing the optical density (OD) of the sample gets you an approximation of the number of cells present. OD measurements are however subject to high variability between labs due to spectrophotometer calibrations and is therefore not a precise approximation method. By instead using the direct method of cell count in each sample, one can remove this source of variability in measurements.
InterLab Study 2018
By comparing how much the following methods agree with each other we can investigate if by using one approach or both, can help to reduce the variability in measurements between different labs.
Conversion between absorbance of cells to absorbance of a known concentration of beads.
By measuring the scattered light from a known concentration of silica beads that are roughly the same size and shape as a normal E.coli cells we converted each lab’s absorbance measurement into a standard “equivalent concentration of beads” measurement
Counting colony-forming units (CFUs) from the sample.
By spreading a known concentration of cells in liquid media on a plate to see how many colonies grow, we can determine the cell concentration of the sample as a whole since each colony should grow from one single cell. We determined the number of CFUs in negative and positive control samples to compute a conversion factor from absorbance to CFU.
Material and Methods
Conversion between absorbance of cells to absorbance of a known concentration of beads.
We made three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve.
Six different parts from the Distribution Kit along with a positive and negative control (all in a pSB1C3 backbone) were transformed into E. coli K-12 DH5-alpha cells. Fluorescence intensity and OD of the samples was measured with same the instrument and standard settings as the calibrations. Measurements were taken after 0 and 6 hours of liquid culture incubation in 37°C and 220 rpm.
Table 1. Parts and Devices used.
| Part | Negative Control | Positive Control | Test Device 1 | Test Device 2 | Test Device 3 | Test Device 4 | Test Device 5 | Test Device 6 |
|---|---|---|---|---|---|---|---|---|
| Part Number | BBa_R0040 | BBa_I20270 | BBa_J364000 | BBa_J364001 | BBa_J364002 | BBa_J364007 | BBa_J364008 | BBa_J364009 |
Figure 1. Fluorescein standard curve based on measured values of fluorescein in serial dilution. Fluorescence intensity was measured with TECAN infinite M200.
Figure 2. Particle standard curve based on measured absorbance of serial diluted samples of silica beads at a wavelength of 600.
Figure 3. Transformation plates from the upper right to lower left: Test Device 4, Colony 2; Test Device 3, Colony 2; Test Device 1, Colony 2; Test Device 2, Colony 2.
Figure 4. Transformation from the right to left: Test Device 5, Colony 2; Test Device 6, Colony 2.
Plates put under UV-light to confirm expression of GFP on all plates before picking colonies for inoculation. One can clearly distinguish a fluorescent expression from the expected cell culture, see figure 3 and figure 4.
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Here is a second paragraph with E.coli written in italics. Any found genes which display promise will have to be validated by qPCR (which is a similar method) in a second run to confirm that they are only expressed due to the strongyle presence. Another approach to tackle our challenge is to screen for interaction between the surface proteins on the strongyle and short peptides. Through affinity screening of a random peptide library displayed on the surface of phages, we can select a peptide with a high affinity to the nematodes surface.