BOX

Aim

This year, the iGEM Team Marburg is participating together with many other teams in the InterLab Study. The aim of the InterLab Study is to investigating the reproducibility of BioBrick characterization by collecting data of the same experiment from iGEM Teams all around the globe. All teams are measuring the fluorescence of three different genetic devices constitutively expressing GFP who differ in their promoter strength. These data will allow a comparison of different protocols and lab techniques regarding the variety of the three parts. We transformed the three devices into E. coli cells, diluted to an optical density (OD) of 0.5 at 600 nm using both, a spectrophotometer and a plate reader and measured the fluorescence with a plate reader. The obtained results showed that the promoter J23101 was the strongest one, the promoter J23106 was weaker and the devices with promoter J23117 showed barely fluorescence over the background.

Design

According to the InterLab Study requirements we measured the three BioBrick parts with promoters of different strengths. The first device consists of the promoter J23101, the GFP coding sequence E0040 and the terminator B0015. The second one is identical to the first device except for the promoter, namely J23106 and the third one uses J23117 as promotor. The promoters are part of a constitutive promoter family that has been introduced to iGEM by the Berkeley Team 2006. The promoters all have the same length and only differ in their sequence. Even small changes have a high impact on the expression level. We used BBa_I20270 with the promoter J23151 as positive control and as negative control BBa_R0040, an empty plasmid with pTetR. The chassis that we used for the characterization was the E.coli strain DH5α. This strain was used as control for the cells’ auto-fluorescence.

Materials and Methods

The parts for the three constructs used in the InterLab study (J23101, J23106, J23117 and I13504,) were taken from the iGEM distribution plates and transformed into NEB turbo cells. Afterwards, the plasmids were recovered through miniprep from an over night culture. The plasmids were amplified via PCR and purified by gel extraction. The final constructs were then assembled using CPEC cloning (Circular Polymerase Extension Cloning). The correct identity of the resulting constructs was confirmed by analytical digest with PvuII and sequencing. The final plasmids were transformed into DH5α cells. For the inoculation and cultivation we followed the recommended InterLab Protocol. We prepared our devices and the controls by streaking them out on agar plates from glycerol stocks. We incubated the plates 18h overnight at 37°C. For that we used LB Agar from Roth without antibiotic for the empty DH5alpha cells and LB Agar from Roth with Chloramphenicol (c=34 ug/ul) for the remaining strains carrying the constructs and controls. We inoculated our strains carrying the devices and controls in 5 mL LB with Chloramphenicol in a 15 mL Test tube. The empty DH5alpha cells were inoculated in 5 mL LB without antibiotic. For each sample we set up three biological replicas. The samples were incubated at 37°C at 300rpm for 16 h.

Measurement

For the plate reader measurement we measured the OD600 of our overnight cultures and therefore diluted them 1:10. As blank we used pure LB plus Chloramphenicol and LB without antibiotic for the empty DH5α cells. We measured our construct strains and the control strains with both the Spectrometer Ultrospec 10 from Amersham Bioscience and the Microplate Reader from Tecan. For the measurement of the fluorescence in the plate reader, the samples were diluted to an OD600 of 0,5 within a deviation of 5%. In order to generate meaningful results of each device, 3 biological and technical replicas were measured.For the extra credit of the InterLab Study we also measured our samples three times, in order to get technical replica.

Results

We measured three different colonies (biological replicas) three times each (technical replicas). Data for each of them are summarized in Table 2.
The fluorescence data show a strong difference between the expression profiles of the promoters. The promoter J23101 shows a 17-fold stronger fluorescence signal than promoter J23106. The weakest promoter is J23117.

Discussion

Using the platereader, we obtained consistent data for the fluorescence intensities. The low error bars indicate that all three biological replica exhibit similar fluorescence levels. We expected decreasing values for the technical replica due to the bleaching effect of repetitive excitation over a short period, but this wasn’t observed here. Unfortunately the positive control exhibited a very low fluorescence, which was almost not visible over the background. In a later study (Measurement), we retransformed the positive control and observed higher fluorescence levels, similar to the ones from construct 2.
Overall, it is great, that the InterLab study has been established within iGEM as this is a great contribution to standardization as well as to characterization of BioBricks. Unfortunately, some aspects haven’t been addressed in the protocol for the InterLab Study a hence remain a variable that may change. It hasn’t been precisely described how to obtain the ODs of each culture. OD is an arbitrary unit and varies between devices. Internally one can generate a correlation between for example the Photometer and the Platereader. But it will remain unclear how the correlation is between different devices in all the labs over the world. This problem could be solved if in the distribution kit there would be a standard OD sample similar to the calibration solution you can buy from photometer distributors. The same can be applied to the fluorescence. Of course, the data can be normalized over the OD and the fluorescence data will then be comparable, in the InterLab Worksheet it wasn’t initially indicated if raw or normalized data should be submitted. An even better normalization could be obtained using an internal fluorescent marker, as we show in the measurement study (link). Also it would be nice to have a transparency data policy and submit the raw data as well. As we have also shown in our Measurement Study, measuring the fluorescence from a diluted stationary phase culture shows a high variation in fluorescence intensities over a population. The lowest variation is during log phase. It could have hence been better to use a different time point for measuring the fluorescence. In order to really establish an InterLab Standard Form, those aspects should have been considered.