Team:Paris Bettencourt/Collaborations

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@iGEM_Paris

Collaborations

The Cell-Free Interlab Study

Cell-free expression platforms allow the production of proteins from DNA without live cells by making use of modified cell lysates. Their application is increasingly popular in synthetic biology where they allow fast iteration of design-build-test cycles of genetic parts and gene circuits. Now is the time to prepare for the widespread adoption of cell-free methods by iGEM teams.

Following into the footsteps of the iGEM interlab study, we prepared a pilot-scale interlab study to test the variation of cell-free expression systems between labs. This was made possible by our generous sponsor Arbor Biosciences, who provided the myTXTL cell-free expression system to our team and others.

Results

Preparation of the cell-free interlab protocol

We selected a GFP expressing plasmid (BBa_I20270) from the iGEM 2018 distribution kit and used pTXTL-p70a-deGFP as an optimized plasmid for high cell-free expression of GFP. We prepared a standard protocol to distribute to participating teams. Our protocol makes use of fluorescein as a fluorescence standard, allowing measurements to be compared across labs.

Distribution of the cell-free interlab protocol

We recruited team GIFU iGEM from Japan to participate in this study with us. Thanks to GIFU iGEM for their patience and great work!

Interlab variation in cell-free expression yields

Figure 1. Comparison between cell-free expression of GFP between Paris_Bettencourt (A) and GIFU (B).GFP fluorescence measurements were made at 0 hour and 15 hours of cell-free expression. The values presented here are normalized to respective fluorescein calibration curves prepared by the teams.

As observed from figure 1 (A) and (B) the high expression plasmid (pTXTL-p70a-deGFP) gives comparatively similar magnitude of fluorescence, indicating a low variability of the cell-free expression system. However we do see a difference in the magnitude of low expression GFP plasmid (BBa_I20270), which could be a result of difference in the way we cloned, isolated and prepared our plasmids.

Conclusion

Future interlab studies should make use of cell-free

Our mini-interlab experiment indicated that cell-free systems can be considered reliable when optimized and could give reproducible results upon following standardized protocols. However, we would propose that more teams participate in this experiment as it is in the case of iGEM-interlab. This would confirm the efficiency of cell-free expression system and the technical variability when executed via various teams with good reproducibility.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
paris-bettencourt-2018@cri-paris.org