Team:Freiburg/InterLab Study
InterlabStudy: Evaluating the Effect of Strains and Temperature
With the InterLab Study, the unique opportunity of collaborating with hundreds of teams all around the world is used. All teams participating in this year's interlab study are asked to measure the fluorescence of certain GFP-encoding BioBricks expressed in host organisms of their choice. To be able to compare the results and to obtain conclusions from the work of the participating students, the experimental protocol (see below) has to be specified and followed precisely.
Introduction
In addition to obtaining general data, we aimed to investigate the direct influence of the different Escherichia coli strains we used for our experiments. As all teams are free to use any host organism of their choice, this has great influence on the study itself. To assess these differences we used a cloning (Top10) and an expression strain (ArcticExpress (DE3)RP) of E. coli. Detailed information on these strains can be found on our strains-overview page. As the ArcticExpress strain is commonly used for heterologous expression of sensitive proteins (e.g plant proteins) at low temperatures, we decided to additionally test two temperature conditions for the two strains during our interlab study experiment. Arctic strains are frequently used with inducible expression systems like the lac-operon in combination with IPTG. Usually the cultures are grown to a certain optical thickness and are then induced. Afterwards the cultures are transferred to 10°C and the expression starts. The BioBricks we got from iGEM were not inducible, so we decided to just split the recommended expression time of 16h into 8h at 37°C for optimal cell mass growth and then transferring the cells to 10°C where they should not increase in mass very much but protein expression should be much better in the case of the Arctic strain than in Top10 cells. For reasons of comparison we also let both E.coli strains grow at 37°C for 16h.
Methodology
The protocol we used, our labjournal as well as the raw-data we obtained can be found on the respective pages.
Cloning and Cell Culture
We used classical cloning with BioBricks to construct the demanded devices and validated the results with a test digest and DNA sequencing. We used BBa_I20270, a GFP expression device in pSB1C3, as positive control and BBa_R0040, a tetracycline resistence in pSB1C3, as negative control. After successfull cloning, chemicompetent cells of both strains were transformed using the plasmids, streaked out on LB-agar plates containing 30µg/mL chloramphenicol and incubated over night at 37°C. From each plate three colonies were picked to inoculate two 5 mL liquid cultures in LB medium with chloramphenicol. This provided us with biological replicates to evaluate the biological variability in our samples. Half of the cultures were incubated for 16 hours at 37°C, the other half was transferred to 10°C after 8 hours.
Measurement
To be able to compare the fluorescence of different samples, the cell density first had to be adjusted to equal levels. This was done by measuring the OD600 of all samples and diluting them so that every sample had an optical density of 0.5 within 5% range. Samples were then pipetted into a 96-well plate and at the same time technical triplicates were prepared for every sample. Fluorescence data were acquired using a plate reader with an excitiation wavelength of 485 nm and a band pass filter of 20 nm and an emission filter of 530 nm with 20 nm bandpass. Comparing the results between different timepoints and different setups (for example in different labs) we decided on converting our data to absolute values using a sodium fluoresceine standard curve (see raw data for more information). Therefore concentrations ranging from 0 to 500 ng/mL were measured each in triplicates, confirming a linear relation between relative fluorescence units and amount of fluoresceine in this range. Using slope and intercept of this curve as well as the absolute volume used (100 µL per well) absolute values for our data could be calculated.
Remarks
Our plasmid's nomenclature differs from the official categorization of the devices as we decided on naming the plasmids with increasing promotor strength - just in opposite to the official nomenclature.
Results
We compared the expression behaviour of two strains of E. coli (ArcticExpress and Top10) under two temperature conditions (8 hours 37°C followed by 8 hours 10°C or 37°C for 16h incubation respectively). In all strains under all conditions the construct pILS1 shows no fluorescence different from the background fluorescence of the negative control. Likewise the fluorescence increased for all strains from the positive control to the construct pILS2, to pILS3. For the positive control the Arctic strain clearly performs better at both temperatures and expresses about twice as good as Top10. But for the remaining two constructs the Arctic strain expresses less or equally good as the respective Top10 cultures. The expression level of pILS2 at 37°C does not differ clearly between both strains whereas at 10°C Top10 cells express more protein. Compared to the positive control the increase is 3-fold for ArcticExpress and 4-fold for Top10 in the first case and nearly 5-fold for Top10 in the second case. The best-performing construct in our study was pILS3 with about 1.5-fold increase in relation to pILS2 for all strains and conditions except Top10 at 37°C where the increase was nearly 2.5-fold. Comparing the strains at 10°C it is not possible to distinguish the expression of both strains, but at 37°C Top10 cells clearly express more protein than ArcticExpress cells. (see figure 1 and 2)
In total the results did not meet our expectations completely, as we supposed for a better performance of the ArcticExpress strain at 10°C. The results observed could be explained such that after 8 hours of growth the cell density for both strains was already at its maximum and although further incubation at 10°C slowed down expression more in case of the Top10 cells the overall difference finally may not be measureable. Additionally we could not induce the ArcticExpress cells, therefore an enhanced expression was not expected.
Overall we could show a strain and temperature dependency of the expression of the InterLab devices. As the choice of the expression host is free for every iGEM team participating in the study, our results suggest the need for a separated evaluation of the results of different strains used.
