Revision as of 03:08, 18 October 2018

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.

Collaboration with iGEM Tübingen 2018 team

In order to test deimmunization tool BERT in the context of a meaningful application, > iGEM Tübingen 2018 team < collaborated with us. Their project focused on the usage of antimicrobial peptides fused together with self-assembling scaffolding proteins as a potential alternative to classical antibiotic treatment.

Dissecting Science

As a pilot team we were honored to collaborate with Marc Santolini in his project “Dissecting Science”. In this project, he aims to study the making and the learning of Science in situ by using the iGEM scientific competition as a model. iGEM presents a unique opportunity to bring together groups of scientific thinkers in a competitive, collaborative, and scientifically rich way. The participating teams share their project and their lab book in open wiki websites.

The study of these wikis has allowed to pinpoint the role of student interactions in the success of teams. Yet, the dynamic nature of these interactions in the lab has remained elusive. How do students collaborate? How are subgroups formed? What is the frequency of interactions with mentors/PIs? How do these interactions lead to better learning (skill spreading), productivity (BioBricks/project size), creativity (project uniqueness) and success in the competition (medals and prizes)? To answer these questions, his team investigated for the first time the interactions in the lab for the our team as a pilot, with the end goal to reach out to a large number of teams. Such interaction was mapped by: 1) using MIT Rhythm badges in collaboration with Oren Lederman and Jordan Reedie from MIT Media Lab; 2) analysis of team interaction through the Gmail, Slack and self-report survey.