Since synthetic biology is built on engineering principles, reproducibility of the experiments and reliability of its standard parts are crucial for building predictable biological machines. This year’s InterLab study required all participants to produce comparable units for measuring the fluorescence of green fluorescent protein (GFP) following the same protocol to test the reproducibility of the experiment. Eight devices encoding GFP under three different promoters as well as two types of ribosome binding sites (RBS) were tested for their reliable gene expression in Escherichia coli.
Materials and Methods
- Eight devices, listed in table 1 inserted in pSB1C3 provided by iGEM Headquarters:
- Expression host: E. coli DH5-α
- Microtiter plate: 96 well plate, transparent with round base, Sarstedt
- Plate reader for fluorescence measurements: Fluoroskan Ascent, Thermo Labsystems
- Plate reader for OD595 measurements: Multiskan ascent, Thermo Electron Corporation
|Test device||Promoter||Promoter strength||RBS|
|Negative control||TetR repressible promoter||medium||-|
All constructs were received in the pSB1C3 plasmid carrying the gene for chloramphenicol resistancy.
The provided InterLab protocol was followed to perform the measurements, as well as the protocol for transformation. Colony PCR, which was not a required part of the InterLab study, was performed using published protocol.
All raw data from measurements can be found here. In order to quantify the fluorescence of the expressed GFP in test devices, a standard curve as in fig. 1, using fluorescein was made. For the absorbance measurements a conversion factor was needed to convert absorbance data to standard OD595 measurements. For that purpose LUDOX-S40 was used as a single reference point. The results from the absorbance and fluorescence measurements are depicted in fig. 3 and fig. 4, respectively.
The result from colony PCR showed the size of the insert was about 1200 bp for the six test devices as well as the positive control device (P), and about 400 bp for the negative control device (N). The negative control contained water instead of the colony PCR product.
After several unsuccessful attempts of obtaining fluorescence readings, colony PCR was performed to ensure the incubated colonies contained the plasmid with the correct gene insert, fig. 2. The gel results of the colony PCR products indicated the correct gene inserts were present, meaning the transformation was successful. Based on the promoter strengths, the following outcomes were expected:
Device 1 and 4 - high fluorescence signal and low OD595
Device 2 and 5 - medium high fluorescence signal and OD595
Device 3 and 6 - low fluorescence signal and high OD595
The relative results matched the expected outcome to a certain extent. However, the absolute values for absorbance and fluorescence were on a level of background noise. Low OD595 values could be explained by low competency of E. coli DH5-α cells. Low fluorescence values might be the due to questionable reliability of RBS and improper physical conditions of the cell. It should be noted that the cell growth on chloramphenicol plates was consistently low throughout the project.