Over the course of the last year we had the invaluable opportunity to meet and work with other iGEM teams from all over the world. Through close collaboration we contributed to their projects, got a lot of feedback and received instrumental help for the realization of a malaria-free world. Apart from various surveys, social media challenges, skype meetings, iGEM meetups and especially the following list of our collaborations we got to know so many dedicated students from all around the world. Thank you!
Bacteria cultures are an established technology for industrial production of complex biological products. Decades of research and development were spent optimizing laboratory and industrial applications designed for highest possible yield. Highly controlled environments were invented: cell shakers and bioreactors provide ideal conditions for bacteria suspension cultures.
In environmental synthetic biology, the situation could not be more different. Cells are supposed to function as designed in uncontrollable environments under varying conditions and no maintenance. If a laboratory cell culture would be left unattended for longer periods of time, cells would overgrow until all substrate is depleted and die. Without shaking, cells would sink to the bottom and form microenvironments with deleterious effects for the desired function.
This issue calls for an innovative solution that allows environmental synthetic biology projects like ours to rely on modified bacteria while still being stable without maintenance. iGEM Eindhoven provides such a solution: They developed a dextran-based gel in which bacteria are immobilized by expression of matrix-adhering membrane proteins. This novel approach of 3D gel-embedded culture ensures even distribution of cells throughout the culture and poses an additional inhibition of bacterial cell growth, complementing our soft growth inhibition Parts. In S.H.I.E.L.D., we employ Eindhoven’s gel as matrix for E. coli. This way, we are confident in sustainability far beyond classic culture technologies could provide in the field.
iGEM Eindhoven developed their gel to be as stable as possible. Despite a desirable attribute for any application, this raises difficulties in characterization: How do you test whether gel-embedded bacteria are still alive and viable? Naturally, you would try to dissolve the gel under conditions embedded bacteria can survive. After their gel proved to be resistant to dextranase, an enzyme specifically for the degradation of dextran, they asked us to try dissolve it under varying conditions.
There are two questions to be answered:
- What conditions are ideal to dissolve the dextran gel while ensuring survival of embedded bacteria?
- If the gel can be dissolved while embedded bacteria stay alive, what are implications for biosafety?
iGEM Eindhoven sent us their gel, and we developed strategies to dissolve it. Eindhoven told us that they were able to dissolve the gel at very low pH, which would not be a feasible environment for bacteria contained within. Based on this information, we set up two moderately acidic conditions: An Aqueous solution of hydrochloric acid at pH 3, at which E. coli is able to survive, but that is not realistic in most environments in which the gel would be used, and a solution of lactic acid at pH 3.61. E. coli produces lactic acid both naturally, and at higher rates in our project2. While we do not think that a pH of 3.6 would occur evenly throughout the entire gel, overproduction of lactic acid may lead to locally raised lactic acid concentrations that come close to our artificial condition. Additionally, we set up two basic conditions: the agarose-dissolving buffer from Thermo Fisher GeneJET Gel Extraction Kit at pH 8, and an aqueous solution of NaOH at pH 9.
We incubated 190 mg gel pieces at 37 °C in 1 mL of aqueous solutions of NaOH, HCl, lactic acid and the Thermofischer GelEx-Kit Binding Buffer for 2 hours and checked for decomposition.
After two hours, the gel piece in aqueous NaOH-solution seems to slowly dissolve as indicated by a decrease of mass (165 mg after 2 h). In aqueous HCl-solution the gel seems to be stable (no change in mass after 2 h). In Thermo Fisher GeneJET Gel Extraction Kit Binding Buffer the gel seems to soak in the solution but not to decompose (215 mg after 2 h). In aqueous lactic acid solution the gel seems to be stable (no change in mass after 2 h).
After 24 h the gel pieces were weighed again. In aqueous NaOH-solution the gel further dissolved as indicated by a decrease of mass to 150 mg. In aqueous HCl solution the gel seems to be dissolving at a slower, but measurable rate to 170 mg. In Thermo Fisher GeneJET Gel Extraction Kit Binding Buffer the gel’s mass increased further to 235 mg. In aqueous lactic acid solution the gel started to slowly dissolve as indicated by a decrease of mass to 157 mg (Fig 1.).
iGEM Eindhoven’s dextran gel is remarkably stable under varying conditions which exceed tolerance of most common bacteria. Given the information that under acidic, non-physiological conditions the dextran gel dissolves, we tested conditions at E. coli tolerance borders. At pH 3 and pH 3.6 in HCl and lactic acid, respectively, degradation was measurable after 24 h of incubation. Since iGEM Eindhoven previously tested their gel’s stability under acidic, but not under basic conditions, we tested degradation at pH 8 in agarose-dissolving buffer, and pH 9 in aqueous NaOH solution. Due to high salt concentrations in the Gel Extraction Kit Buffer, the gel’s density increased over time, rendering a possible degradation impossible to measure. However, visual inspection indicated that the gel was intact after 24 h of incubation. Under more basic conditions, the gel dissolved. While at pH 8, E. coli is able to survive, we expect E. coli to be disrupted when incubated at pH 9 for 24 h.Visit Eindhoven
We are very grateful to get in contact with iGEM team Marburg and want to thank them for organizing an amazing German iGEM meetup. Additionally, they helped us with our Golden Gate assembly by sending us an alternative protocol for our assemblies. With great care we participated in their well-organized InterLab Study using Vibrio natriegens, for developing a new model organism for prokaryotic cloning. For this we repeated the InterLab protocol they optimized for Vibrio natriegens.
Why is working with Vibrio natriegens not only good for the iGEM Team Marburg, but also for our approach and almost all synthetic cell biologists?
When you are familiar with microorganisms and talks about Vibrio genus, you may think that “it is an ordinary halophile organism”, but what you actually should think is “Well, may this little super organism can change the whole cloning workflow all around the world”!
This organism has a doubling time of under 10 minutes. It can be carried out easily in your lab work as a nonpathogenic, fast growing prokaryotic organism with a broad range of pH tolerance.
The iGEM Team Marburg wants to Establish Vibrio natriegens as a new chassis organism for synthetic biology. They designed three new strain, which make work in the lab much easier and more effective. Imagine you just need one day for a whole cloning process. If this bacterium were be establish last year, we (and we think almost every iGEM team) could achieve 3 times as much since everything works three times as fast as in Vibrio natriegens. We did the InterLab study to help them getting done with their establishment. We transformed the included devices in Vibrio natriegens and measured the growth with a plate reader. We hope they will be succsesfull and wishes them all the best.Visit Marburg
The iGEM Team Makerere University helped a lot by personally evaluating and discussing our project and its application in countries like Uganda. They distributed our questionnaire to people working with malaria on a daily basis. Thanks to them we got invaluable expertise and input from social workers, nurses, lab technicians and epidemiologists. In return we assisted them with the planning of their experiments concerning the expression of PETase and proposed a basic kinetic cell model for their project.Visit Makerere
We would like to thank iGEM Team Düsseldorf for organizing the iGEM postcard exchange. This enabled us to get to know various other iGEM teams with fascinating projects via well designed cards. Moreover, we really appreciate their commitment in providing us the theoretical knowledge to work with cyanobacteria. Finally, we are grateful for kindly allocating a magnificent picture of their cyanobacteria for the use in our wiki.Visit Düsseldorf
We participated in IIT-Madras Language Project. They produced educational videos about synthetic biology for their YouTube channel in various languages. We translated their English script to German and Polish, recorded the audio in these languages and they produced the finished videos.Visit IIT-Madras
- ↑Small, P., Blankenhorn, D., Welty, D., Zinser, E. & Slonczewski, J. L. Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J. Bacteriol. 176, 1729–37 (1994).
- ↑Bunch, P. K., Mat-Jan, F., Lee, N. & Clark, D. P. The IdhA Gene Encoding the Fermentative Lactate Dehydrogenase of Escherichia Coli. Microbiology 143, 187–195 (1997).