Team:Lund/Contribution
Lab Contribution
We made several contributions for future iGEM teams within the scope of the project. Before the lab work started, we invested much time in literature research. We gathered valuable information about engineered probiotics and suitable signal peptides for L. reuteri and E. coli. Furthermore, we researched suitable shuttle vectors. We hope that the gathered information will help future iGEM teams designing an engineered probiotic.
In the first cycle of the project, the lab team optimized the ThT assay for curli detection. Future iGEM teams who would like to do similar amyloid detection could use our refined assay. We experienced that pollution in the extracted plasmid usually is caused by either genomic DNA contamination or RNA pollution during plasmid prep. We could reduce the risk of contamination by gently handling the lysate after adding buffers P2 and P3 to prevent shearing of chromosomal DNA and checking the shelf life of RNase included in the kit before adding. Furthermore, when doing plasmid preparation, we were able to optimize the antibiotic concentration.
We realized that either a too high or low antibiotic concentration resulted in a low plasmid yield. The optimized concentration of Erythromycin was 10 µg/ml. We hope that this concentration value will give future iGEMs teams a feel of concentrating their antibiotics when doing a plasmid preparation. In the first two cycles, we used Erythromycin as a selection marker. However, during the lab work, we realized that the choice of Erythromycin was insufficient since some E. coli strains can be naturally resistant to it. In addition, the concentration of antibiotics used for selection after ligation and transformation depends on both the competent cells and vectors and a relatively low concentration can cause false-positive colonies. We hope that future teams will learn from our experience and will choose their selection marker and concentration of antibiotics carefully.
During cycles one and two, we were trying to assemble the plasmid and inserts via Gibson assembly. After we failed to use this method, we switched to restriction enzyme digestion with subsequent ligation. We were then able to obtain a successful transformation. From our experience, restriction enzyme ligation has a higher likelihood of a successful transformation. Lastly, the selection of the position of inserts in vectors is crucial for engineering success. During the first cycle, the position of the inserts was in the middle of the previous GFP construct, which may have caused both transcribed and translated short GFP RNA strands and our inhibitor protein. It is better to remove the inserts from the vector before engineering.
Human Practice Contribution
The Human Practice contribution consists of a Workshop Guide. The main aim of developing a Workshop Guide is that other institutions, specifically iGEM teams, can employ it to design exciting workshops related to their projects. The workshops will help educate the general population about various scientific topics related to synthetic biology, sorting out the misinformation issues. By extension, popular opinion will be more based on science, and it will have more relevance. To read more about our Workshop Guide and other educational efforts, visit our Education & Communication page.
Modelling Contribution
Another significant contribution to the future iGEM teams is the Inhibitor model. Building on previous models by other iGEM teams, it is currently the most complex of its kind by an iGEM team. The model is also designed to be easily extended for a multitude of inhibitor-protein interactions. For more information, visit our modeling page.