Interlab measurement study

Reproducibility is one of the most crucial parts of reliable research. However, lab does not equal lab and reproducing data achieved in one lab might not be achieved in the same way and extend in another lab around the world. The iGEM 2015 Measurement Interlab Study addresses the question how consistent research results are in a global context. All participating teams in the Measurement Interlab Study have been given the same task to construct three promotor-GFP devices and measure their fluorescence.

We assembled all three constructs in the high-copy number plasmid pSB1C3 and transformed E.Coli TOP10 cells to express GFP signal. The only difference between the constructs is the promoter used to drive the GFP expression. We measured the GFP signal at an OD of approximately 0,6 in a 96-well plate. The obtained results showed a broad variability of signals within one construct, most probably due to impurities on the colony plate. However, we can see that the device 1 consisting of the biobricks J23101 and I13504 are expressing by far the highest signal. We also see that device 2 (J23106 + I13504) show the second strongest signal, even though only twice as strong as device 3 (J23117 + I13504) which we would have expected to be 10-fold stronger when comparing with data given in the iGEM registry and the strength of each single promotor.

Experimental design

For the Interlab Study, we were supposed to use four different biobricks, the three promotors (J23101, J23106 and J23117) which we should each single one of them combine with a GFP sequence downstream of the promotor. For each single part we extracted purified biobricks from the distribution kit. Using the 3A assembly method (following the iGEM protocol), we constructed all three devices in the high-copy number plasmid pSB1C3 and transformed them into E.Coli TOP10 cells.

Each colony selected has been prior analyzed by gel digestion with AvrII and PstI to confirm the presence of the promotor and by the fragment size the present of the GFP in the target plasmid. After confirming the strains, we restreaked them on fresh LB-agar plates and cultured them until measurement.

After constructing all devices, we grew then in severated E-flasks containing LB medium and chloramphenicol. We grew them to an OD of approximately 0,6 and meaured their fluorescence.

As a control we used only pure LB-medium to substract the fluorescent coming from the medium. In retrospect, it would have been also valuable to have a proper non-fluorescent bacteria as control.

Materials and Methods

Constructs and Strains

All transformations for this study have been performed in E.Coli TOP10 cells. All biobricks were isolated from the DNA Distribution Kit and transformed in order to amplify the plasmids which contain the genetic elements necessary for our device assembly. The chemocompetent cells were consequently transformed by Heat-Shocking at 42°C using a 5x KCM buffer to competence and transformation efficiency.

The amplified plasmids were then recovered using the QIAprep Spin Miniprep Kit (QIAGEN). In a next step, we followed the 3A assembly protocol suggested from iGEM in order to create the devices. Therefore, we cut the promotor parts (J23101, J23106, J23117) with EcoRI and SpeI whereas the GFP sequence (I13504) has been cut with XbaI and PstI. After ligation, we used 2uL of the ligation product for transformation. All constructs were plated on chloramphenicol plates.

Subsequently, colonies were picked at the next days and analysed by digestion with AvrII, a promotor specific restriction enzyme, and PstI. As the GFP sequence is rather long with approximately 900 bp, we can see by size of the fragments whether GFP has been inserted. Furthermore, the specific cleavage of AvrII within the promotor sequence will give us indication of the presence of the promotor. Sequencing could unfortunately not be applied for any biological clone.

Inoculation and Cultivation

Strains were cultured in 250mL E-flasks filled with 10mL LB medium and the corresponding antibiotic (here: Chloramphenicol 25µg/mL taken from a 1000x stock solution). The cultures were kept at 37°C and incubated at 150 rpm.

Precultures were taken from LB-agar plates and incubated overnight at 37°C under permanent agitation. Using a photospectrometer, the OD was permanently measured.

GFP measurement

All strains were picked from LB-agar plates and left for overnight in separated 250mL E-flask in order to let them grow until they reach an OD > 1. We diluted the sample then to an OD of approximately 0,6. For the OD measurements, an Eppendorf (______) was used. When the sample has been diluted to the right OD, we havest cells by centrifuging 1mL of culture for 5min, 13.000 rpm. We removed the supernatant and resuspended the pellet was subsequently resuspended in 1X PBS. Each sample was applied in technical and biological triplicates (expect of device 3 were we could find only one positive clone after cloning). The fluorescent was measured using a Clariostar (BMG LABTECH) and analysed using MARS Data Analysis software.

Statistical Analysis

Signal was corrected against the medium blank and then normalized by the corresponding OD. For each technical triplicate an average has been calculated. Subsequently, we took the mean of the three means describing the three biological replicates (for device 1 and 2; one biological replicate for device 3) in order to get a value for each construct. The standard deviation has been calculated from all three means representing the biological triplicate. Statistical significance was calculated by Students T-Test.

Results and Discussion

As sequencing was unfortunately no option for our constructs, we verified our assembly by digestion using the promotor-specific AvrII and PstI. This digestion give us a two fragment of a size of 920 bp and 2068bp.

We have seen the proper sizes for all constructs tested. However due to time constraints, we were unable to find the last two replicates for the device 3. After 16-hours incubtion, the GFP signal was measured and normalized by the sample OD.

As displayed in the graph above, we see quite some strong fluorescence coming from GFP which is however mostly due to one biological replicate explaining the huge standard error of this bar. Device 2 about two-times as strong as device 3 and therefore the order or devices is following their pre-described strenghts (REFERENCE: iGEM Promotor Strength table). In comparison to the documented promotor strengths, the difference between device 2 and 3 is still too small as a ten-fold difference would have been expected. One of the reasons for this small difference might be that device 3 contains only one data point as only one biological replicate could be produced. Due to huge error bars, it is however impossible to conclude from this data.

In order to produce a more reliable data set, we would have needed to re-investigate our strains used and double check their purity. Furthermore, we would need to aquire the last two replicates for device 3 and reperforom our experiments.