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InterlabStudy: evaluating the effect of strains and temperature

With the InterLab Study the unique possibility of collaborating with hundrets of teams all around the world is used. All teams participating are demanded 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 results from the work of the participating students, the experimental protocol (see below) has to be spcified and followed precisely.

Introduction

Besides obtaining data for the common good our experiments were aimed at the direct influence of the strains used. As all teams are free to use any host organism this has great influence on the study itself. To asses these differences we used a cloning (Top10) and an expression strain (ArcticExpress (DE3)RP) of Escherichia coli. Detailed information on these strains can be found on our strains-overview. As the arctic strain is commonly used for heterologous expression of sensitive proteins (as plant proteins) at low temperatures we decided to additionally test two temperature conditions for the two strains. Arctic strains are frequently used with inducible expression systems as the lac-operon in combination with IPTG. Normally the cultures grow to a certain optical thickness and then are induced. Afterwards the cultures are transferred to 10°C and the expression is started. The BioBricks we got from iGEM were not induceable, 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 shouldn't increase in mass very much but protein expression should be much better in the case of the Arctic strain than in Top10 cells. For comparison reason we also let the two E.coli strains grow at 37°C for 16h.

Methodology

The protocol we used, the InterLab worksheet as well as our results 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 DNA sequencing as well as restrition mapping. 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 with the plasmids, stricken out on LB-agar plates containining 30µg/mL chloramphenicol and incubated over night at 37°C. From each plate three colonies were picked to inoculate two times 5mL liquid cultures in LB medium with chloramphenicol This provided us with biological replicates to asses the biologcal variability in our samples. Half 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 had first to be equalized. This was done by measuring the OD₆₀₀ of all samples and diluting them so that every samples lay within 5% range of an OD₆₀₀ of 0.5. Samples were then pipetted into a 96-well plate whereas technicel triplicates were prepared for every sample. Fluorescence data were then acquired by using a plate reader with an excitiation wavelength of 485 nm and a band pass filter of 20nm 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 measures using a sodium fluoresceine standard curve (see interlab_results). Therefore concentrations ranging from 0 to 500 ng/mL were measured in triplicates, confirming a linear relation between relative fluorescence units and amount of fluoresceine in this range. With slope and intercept of this curve absolute measures for our data could be computed.

remarks

Our labeling of the plasmids differs from the official numbering of the devices insofar as we decided on naming the plasmids with increasing promotor strength - just in opposite to the official nomenclature.

our construct	BBa	expected promotor strength
pILS 1	J23117	162
pILS 2	J23106	1185
pILS 3	J23101	1791
positive	J23151 1bp mutant from J23114	256 (J23114)

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 under at both temperatures and expresses about twice as good as Top10. But for the remaining two constructs Arctic strains express less or equally good as the respective Top10 cultures. The expression level of pILS2 at 37°C does not differ clearly between bot strains whereas at 10°C Top10 cells express more protein. Compared to the positive control the increase is 3-fold for Arctic 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 disinguish the expression of both strains, but at 37°C Top10 cells clearly express more protein than Arctic cells.

Fluorescence obtained for different strains of E. coli at 10°C with different constructs. The blue area depicts measures of the negative control plus three times its standard deviation; results in this area are not different from the background and thus are considered to be zero.

Fluorescence obtained for different strains of E. coli at 37°C with different constructs.

In total the results did not meet our expectations completely, as we hoped for a better performance of the Arctic 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 further incubation at although further incubation at 10°C slowed down expression more in case of the Top10 cells the overall difference finally may not be measureable.

So in total we could show a strain and temperature dependence 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.

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