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In the spirit of iGEM, we have made several contributions to the futures iGEM teams:

InterLab 2022 Study

This year our team has been inspired to participate in the InterLab study due to the echoes received from iGEM Evry Paris-Saclay teams that participated in previous editions and from teams that did not get the chance to be involved in such an enriching experience. InterLab benefits participants by setting the ground for the implementation of the biological techniques needed throughout the development of the project. Additionally, the participation in this study improves communication among team members, a paramount aspect for increasing productivity, collaboration, organization, and robustness of downstream analysis.

In the beginning, we wanted to perform all three experiments indicated on the form. However, for unexplained reasons we were unable to recover half of the devices required for Experiment 2, even though we tried transforming E. coli several times with both homemade and commercial competent cells. Thus, we only submitted results for Experiments 1 and 3 as indicated, and these results are also available on this page.

The calibration experiments are detailed in the Measurement page of the wiki, along with other calibrations that we performed for our other types of experiments.

We are fascinated by the experience and eager to learn more about the worldwide InterLab results.

Experiment 1

“This experiment aims to assess the lab-to-lab reproducibility of the new three color calibration protocol. We will test if it works well for calibrating the fluorescence in cells that express one single fluorescent protein and for cells expressing two different fluorescent proteins at the same time.”

We tested 8 different devices in bacteria E. coli DH5ɑ in order to measure the expression of 4 fluorescent proteins (GFP, mRFP1, mCherry & BFP) by using different terminators (B0015, rnpB T1 & rpoC T), the B0032 ribosome binding site (RBS) and the J23101 promoter, except for the positive control where the J23151 promoter was used. Some of these devices also included the genetic insulator RiboJ for its potential positive effect on the expression of the downstream gene (Figure 1).

Figure 1. InterLab 2022 devices used for Experiment 1.

We performed the Experiment 1 according to the provided protocol, using a CLARIOstar (BMGLabtech) plate reader and a opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). Our submitted results are available in the excel file.

Moreover, we took advantage that the CLARIOstar (BMGLabtech) plate reader was equipped with shaking and incubation features and continued monitoring the OD600, and the green, red and blue fluorescence overnight for an additional 14 hours. Fluorescence values were normalized by OD600. Furthermore, the arbitrary units have been converted into Molecules of Equivalent FLuorescein (MEFL) / particle (more details about the calibration curves on the Measurement page of this wiki).

Initial recordings at time 0h has revealed high intensity of fluorescence among all constructions tested, that was no longer present during consecutive recording at 6h. Importantly, the highest among the devices were seen for Test Devices 5 and 6. This high Fluorescence/OD600 values are mathematical artifacts, as the initial OD600 values were very low.

Positive control of the experiment, carrying GFP showed as expected a steady increase of green fluorescence, maxing out at 20h after the experiment. Test Device 1, although carrying the same fluorescent protein, demonstrated a lower expression of GFP when compared to the positive control. The only difference between the two devices is the promoter. J23101 is considered a rather strong promoter according to the documentation available on its page of Parts Registry, while J23151 is a one base mutant of J23114 which is a lower strength promoter than J23101. Our results suggest that this mutation strongly increases its strength.

Devices expressing mRFP1 and mCherry, two red fluorescent proteins, remained initially inactive. At 20h, Test Device 3 emits almost the double signal of the mRFP1 counterpart (Test Device 2). Interestingly, the Test Device 4, carrying mCherry as Test Device 3, but with RiboJ has an expression intensity lower than Test Device 3, comparable to that of Test Device 1.

Finally, the Test Device 5 and 6, both carrying the same promoters, same RBSes and the same insulator in the same order, but are different in the fluorescent proteins. Test Device 5 expresses BFP followed by mCherry, but preferentially expresses BFP, while mCherry expression is negligible. On the other hand, Test Device 6 has exhibited similar expression of both GFP and BFP proteins that it carried. Although Test Device 6 expression of both proteins were orders of magnitude lower to that of Test Device 5, with a disparity or preference of BFP expression over GFP. Expression profiles of Test Device 5 and 6 can not be attributed to the RiboJ role, since we didn’t have a control for a device which contained two distinct proteins without an insulator. Although, it is interesting that in either Test Device 5 and 6, expression intensity was higher for BFP. Here it is important to notice that all samples pertained to a certain noise of blue fluorescence.

To conclude, both positive and negative controls have performed as expected. We were able to detect the expression of each fluorescent protein either when expressed individually or two by two.

Figure 2. In vivo characterization of fluorescent proteins expression by E. coli DH5ɑ cells carrying the InterLab 2022 Devices (Figure 1). The data and error bars are the mean and standard deviation of four measurements on independent biological replicates.

Experiment 3

This experiment aims “to test protocols that will eventually be automated. For this reason, we will use 96-well plates instead of test tubes for culturing. Consequently, we want to evaluate how the performance of our plate culturing protocol compares to culturing in test tubes (e.g. 50mL falcon tube) on a global scale.” .

We performed Experiment 3 according to the provided protocol, using a CLARIOstar (BMGLabtech) plate reader and a opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). Our submitted results are available here as an excel file.

As in the case of Experiment 1, we took advantage that the CLARIOstar (BMGLabtech) plate reader is equipped with shaking and incubation features and continued monitoring the OD600 and the green fluorescence of plate 2 overnight for an additional 14 hours.

The 8 individual devices of Experiment 3 (Figure 3) contain 7 different promoters (J23101, J23151, J23106, J23117, J23100, J23104 & J23116) which have different strengths (Table 1). In addition to the promoter, the expression of the GFP gene is modulated by either the B0032 ribosome binding site (RBS) in the case of the controls or the B0034 for the test devices, the B0015 terminator and a RiboJ to insulate translation in the negative control device.

Our results (Figure 4) show, as in the case of Experiment 1, a general decrease of the fluorescence over time until 6h for all the test devices, which is an expected mathematical result.

At 6h, one can notice a slight difference between Plate 1 and Plate 2 for all devices. Plate 1 was incubated in the plate reader, while Plate 2 contains the bacteria grown in tubes over the same period. This is a strong support in favor of automatisation and miniaturization of such experiments.

At 20h, we observe an important fluorescence signal for the majority the devices and the positive control, except for the Test Devices 3 and 5. For Test Device 1, 2 and 4, the obtained signal at time=20h is even higher than the positive control.

Here, it must be noted, that the promoter strength to signal ratio was mostly followed at anticipated increase. For example, Test Device 4 has the strongest promoter (strength 1.0) and gave the highest recorded fluorescence, while Test Device 3 exhibits the lowest fluorescence and carries the weakest promoter of them all (strength 0.06). This logic applies to almost all samples pairs, other than Test Device 1 and 5 where, for a very similar promoter strength the GFP expression level is very different.

To conclude, it is now clearly established that the correct choice of promoter used for the expression of a pathway is a major determinant of the success of the associated application. Here one must note that not only are the strong promoters desirable, but for specific applications, weaker promoters may also be preferred.

Figure 3. InterLab 2022 Devices used for Experiment 3.

Table 1. Strengths of the promoters used in the test devices of Experiment 3, according to data available in Parts Registry.
Part name Strength
J23101 0.70
J23106 0.47
J23117 0.06
J23100 1.00
J23104 0.72
J23116 0.16

Figure 4. In vivo characterization of GFP expression by E. coli DH5ɑ cells harboring the InterLab 2022 Devices (Figure 3). The data and error bars are the mean and standard deviation of four measurements on independent biological replicates.

3D printed models for MFC

We made two microbial fuel cell designs for FMD-3D printing (big and a small one). In the Figure 5 larger MFC model and its safety box are visualized, as they are observed when they are assembled. For full model descriptions and design instructions please refer to our hardware chapter.

Figure 5. Large MFC assembly, as observed in the CAD software Onshape. This figure does not include bolts and nuts that were used for sealing the MFC chambers.

New documentation to existing Part Pages on the Registry

We turned to Parts Registry for parts built by previous iGEM teams that might be useful for our project. We found and used a few of them, as can be noticed both on our wiki and on the design of our composite parts.

While most of them had an appropriate description, some of them were lakning it. To bring our contribution for future iGEM teams we added documentation to BBa_J64822, BBa_K3257001 and BBa_K4289009.