Team:EPFL/HP/Gold Integrated
Integrated Human Practices
Educational Cell-Free Mini-Kit
The beginning of the Educational Cell-Free Mini-Kit
Throughout our project we were in contact with professionals from a broad range of disciplines. We took part in a one-day workshop to integrate cell-free biology in high school curricula. As a result, a complete ‘toolbox’, the Educational Cell-Free Mini-Kit, that swiss high school teachers could include into their classes, was developed. However, we have no knowledge and experience in curriculum organisation which is why we got in touch with several high school teachers. We introduced them to our project and our experimental mini-kit and asked them for feedback on how it could be improved. Based on their opinion we adapted and changed the Educational Cell-Free Mini-Kit (ECFK) so that it eventually could be included in the official high school curriculum in biology.
"Votre projet me paraît passionnant et je serais très heureux de parler avec vous des implications et applications dans les classes. Il pourrait faire l'objet d'une publication dans le cadre du projet Bio-Tremplins de la faculté des sciences et du DIP Genève qui s'adresse aux enseignants et touche près de 400 personnes à travers la francophonie."
"Your project seems fascinating to me and I would be happy to talk to you about implications and applications in class rooms. It could be part of a publication in line with the project Bio-Tremplins of the science faculty of the university of Geneva that is addressed to teachers and will touch up to 400 people all over the french speaking part of Switzerland."
"Je parle chaque année du projet iGEM à mes élèves de 4OS, alors si pour une fois il y a moyen de faire plus que de regarder une vidéo, je serai ravi de le faire."
"Every year I talk about the iGEM project to my last year high schools students, hence if, for once, it is possible to do more than watching a video, I would be happy to do it."
"Quel projet captivant et motivant sur lequel vous travaillez ! Il est toujours sympathique d’avoir des nouvelles d’anciens élèves et d’être informés de ce qui se fait dans les laboratoires. Nous sommes intéressés par votre kit."
"What a captivating and motivating project on which you are working ! It is always nice to hear from former students and learn more about what is being done in laboratories. We are interested in your kit."
The safety side
After having a more detailed idea on what the Cell-Free Mini-Kit will look like, we reached out to EPFL's biosafety department to make sure we were complying with national safety requirements. Stéphane Karlen, the main coordinator for biological safety on campus, made a risk evaluation of the kit and concluded that the Cell-Free Educational Mini-Kit does not represent a risk for the user or the environment.
A kit ? Cool idea, but in reality ?
The Cell-Free Mini-Kit consists of several tubes containing different lysate reaction components, ideal to show a range of biomolecular processes upon rehydration. At the same time it is equipped with a detailed description of the experiments that could be performed with it as well as suggestions as to which topics a biology teacher could cover with them. The Cell-Free Educational Mini-Kit is a diverse toolbox suitable for demonstrating biological principles to all age groups, as the teacher is free to provide the students with an adequate background for their grade level.
The Cell-Free Mini-Kit includes the material and experimental setup for the following four questions :
- Presence of the ß-galactosidase enzyme in a cell used for lysate production
- Expression of protein in a cell-free extract
- Interest of energy supply for biochemical reactions
- Polymerase operating mode
- Repression of a gene
The experiments will include the following procedures :
- Detection of ß-galactosidase enzyme presence in a lysate made of cell culture by colorimetric assay
- Study of cell-free in vitro expression of ß-galactosidase and kinetics of ß-galactosidase activity
- Showing the importance of an energy source by performing cell-free experiments with and without energy solution
- Testing different polymerases in cell-free environment with different promoters
- Study the principle of gene repression using a riboswitch which can be expressed in the presence of a trigger
Components for the abovementioned reactions include :
- E.Coli cell lysate of BL21, DH5alpha and M15 strains
- Energy solution, a mixture containing amino acids, salts, reducing agents
- Buffer A, contains Tris, magnesium and potassium glutamate
- Substrate, chlorophenol Red-β-D-galactopyranoside
- Linear DNA templates to be expressed in vitro (for example LacZ)
- Our toehold/trigger construct, which is single and double stranded DNA templates
The kit exists in two versions: either all the materials are freeze-dried and delivered in tubes containing the separate freeze-dried components, or be distributed in their fluid form using dry ice.
What happens after the Jamboree ?
The next issue we had to settle was how to supply the schools with the components needed for the Mini-Kit, in a sustainable way that will last longer than just the duration of this year's iGEM project. It was clear that after November 2017, our team would not have access to the lab spaces anymore and we would therefore not be able to deliver components of the Cell-Free Mini-Kit ourselves. We looked into more details for the following two solutions:
- Having one of the labs at EPFL produce the lysate and energy solution (for example within the scope of the laboratory initiation course for second year students at the faculty of Life Sciences)
- Contacting the company responsible for delivering laboratory material to the high schools directly
It was the second point that caught our attention after the meeting with the teachers. They gave us the contacts of BiOutils (french for bio-tools), whose mission it is to support the teaching of modern biology at local high schools and supply them with the material and competences needed. We have established a collaboration with BiOutils: we are at the moment in discussion with them and exchanging the protocols for lysate production and energy solution, to see when and how it would be possible for them to produce all the components of the Cell-Free Mini-Kit on a larger scale and supply the high schools directly once the iGEM competition will be over. This solution is definitely the most satisfying as the Mini-Kit will enter a list of supported cantonal experiments for high schools and biology teachers in all of Geneva would be able to order it and use it in their class.
We are very glad to have found such broad support and interest in the idea of our Cell-Free Educational Mini-Kit and hope that it will become permanently integrated in local high school curricula. With the help of BiOutils, the Mini-Kit would be produced on a larger scale and will be accessible for every high school in the region. Its future use will be ensured even after the Jamboree and will create a long lasting impact on education.
How do you asure that it will be appropriate for students ?
Our interactions with the high school teachers were very valuable in order for us to
gather opinions and reactions towards the Mini-Kit we wanted to propose. After exchanging
the protocols with the teachers from Collège Calvin, they suggested to us that
lyophilisation of the components would not be needed, as they were equipped to
directly manipulate the solutions. They told us that it would be better for the
course since the students would be the ones pipetting the solutions and preparing
the mixtures and that it would make the Mini-Kit more interactive. We of course
agree with this statement and went on to modify our Cell-Free Educational Mini-Kit
protocols to include manipulation of the solutions directly and describe which amounts of reagents would be needed for delivery. Hence, after this discussion, we now propose two different kits: one lyophilised, to facilitate storage in high schools lacking the necessary facilities, and one non lyophilised, for high schools that have access to pipettes and adequate storage units.
We managed to include many other aspects of the teachers' feedback into our Minikit, although two of them persist until now: The central questions of each experiment might be a bit too easy for high school students and it would be great to have two versions of the instructions, one for the students and one for the teacher.
After the visit, the two teachers told us they were very eager to try out a test kit such as to integrate the kit before the end of the ongoing semester in February. We promised to provide them with five test kits as soon as we come back from the Giant Jamboree in Boston so that they could get started with experiments!
The company BiOutils wanted to know how the individual components could be synthesized or produced, as they would be taking over this part for the high schools. We quickly provided them with our detailed protocols on how to make lysate, which cell lines are optimal for cell-free protein expression, how to prepare the energy solution and which substrates and buffers are needed for the reactions.
After an hour-long conversation and several e-mails exchanged with the École Moserin Geneva, we had established that École Moser would like to include the Cell-Free Mini-Kit in their curriculum but could only do so once the next academic year begins (starting Summer 2018). They are also being supplied by BiOutils and asked us if it were possible to expand the Mini-Kit with certain experiments for more advanced biology courses. This suggestion was very welcome and we set out to write a manual that may serve as a road-map for generating new Mini-Kit experiments. At the moment we are discussing which experiments would be most useful and how to realize those with the Mini-Kit.
Choice of viral strain
Even the best biosensor is worthless if the component it is detecting does not have a considerable impact. Scientists in the field of viral infections and immunology helped us figure out several viral strains we could consider for our project.
"I may recommend you to test the following “common” viruses: for DNA viruses B19 (ssDNA virus) or Hepatitis B and for RNA viruses Hepatitis C or human pegivirus 1 (HPgV1)." Samuel Cordey
Reaching out to industry
Our project was confined to the EPFL campus and we wanted to reach more people outside of the academic world. To figure out how we could improve our project further and whether our concept would be interesting to industry labs, we contacted Medisupport, a laboratory group that performs a broad range of medical analyses for hospitals and doctors' offices, servicing Switzerland in it's entirety. As they are in close contact with the industry, they have a different opinion and they would know exactly how Aptasense could be implemented and what were its strengths and weaknesses. They turned out to be quite interested in our project and our protein detection system, consequently, in October one of their representatives came to EPFL to meet with one of our team members and discuss our diagnostics tool in greater detail. After two hours of discussion, Medisupport told us they would want to put us in contact with one of their geneticists. Hence, the following week, we had a second meeting and they gave us more details about the project's outlook and how it could be improved.
They found the general idea quite ambitious and new, which was one of our goals. However, as they understand the world of diagnostics well they told us that it is extremely difficult to implement a new tool because of the trust that we must have in the results given by the diagnostic tool. Hence, it requires a vast series of tests that we couldn’t achieve considering the time and ressources available. Thus, one anther field to reach was the food industry as the testing methods are less developped. Concerning the diagnostic field, Aptasense could have a future in liquid biopsy for detecting tumoral DNA in blood or detecting bacterial markers/activity, considering that the methods used nowadays are quite expensive. In conclusion, it was a great opportunity to meet with them and to have the possibility to implement their advices.
