DIYbio

Overview | Concept | DIYbio | Workshops | Exhibition | Evaluation | Conclusion

Public biobrick:[http://partsregistry.org/Part:BBa_K729016 BBa_K729016: Antifreeze protein, type I from Oceanibulbus indolifex HEL-45]

Sequence: http://www.ebi.ac.uk/ena/data/view/EDQ05862

Planning

Prior to the workshops, we visited the Hackspace on several occasions to prepare for the wetlab sessions. Our visits included giving a short introduction to synthetic biology and our project, a large health and safety discussion, and a primer design workshop. From our early interactions with the citizen scientists, we found that they had previously performed many PCRs and gels. Some of these had been successful. However they have not attempted cloning before, due to the of lack laboratory license and UK regulations. What they wanted from this collaboration was to visit our lab to see how we conduct experiments in an academic institution. They also wanted to perform all the steps in the cloning process. Therefore in the UCL workshop schedule, we attempted to fit all the cloning experiments in three days.

Selecting genes to PCR from Oceanibulbus Indolifex

Which genes to PCR? The Biohackers researched a list of genes from the [http://www.xbase.ac.uk/genome/oceanibulbus-indolifex-hel-45/NZ_ABID01000001/features Oceanibulbus Indolifex genome database] and selected genes which they thought would be useful when transformed into E.coli. There were a number of potential candidates, including metal binding proteins, arsenic reductase, mercuric reductase and antifreeze. The list was then shortened to antifreeze and mercuric reductase. Antifreeze was chosen because a relatively easy assay was found to characterize the gene, while mercuric reductase was chosen due to its useful ability to detoxify mercury.

Hackspace Workshop 29th-31st

Hackspace Wetlab Workshop schedule

In the Hackspace, we worked with the Biohackers on making competent E.coli (W3110) cells in the lab. This was an exciting experience, as we had to make a DIY incubator and shaker ourselves. We had help from many people in the Hackspace from sawing wood to circuit building. For the PCR, we autoclaved our boxes of tubes and tips using their makeshift autoclave (a pressure cooker) and then the boxes were put in the oven to dry.

UCL Workshops 3rd-5th September

Original UCL Wetlab Workshop schedule

In the beginning of September, we held a three day workshop in UCL's teaching laboratory for the citizen scientists. We had an ambitious plan for the workshops, from genomic DNA extraction to ligation and transformation. This was a packed schedule. However, after day one, we quickly realised that it would be a much better learning experience for the Biohackers if the pace was slowed down. The amount of practical experience of each citizen scientists also varied. We allowed much longer time intervals between the practicals to answer the Biohackers' questions and to show them professional techniques we use in the laboratory.

Unfortunately, the first genomic DNA extraction was unsuccessful and some planned workshop items were delayed. However, the participants remarked how exciting and insightful they found the trouble-shooting that ensued.

Post-Workshops

Some of the work was done after the workshops. Now that the Biohackers have the experience of working in an academic laboratory, they pay more attention to reducing contamination, and health & safety, particularly with regards to Ethidium Bromide.

Write-up and results analysis

One of our aims for the collaboration was to encourage note-taking to track errors and improve the accuracy of the experiments ran in the London Hackspace. The Biohackers kept their own lab-books for the duration of the workshops and uploaded this data to their wiki.

Primers, protocols and results can be found on the [http://wiki.london.hackspace.org.uk/view/Public_Biobrick#Visualisation_and_interpretation London Hackspace Wiki]

"[Some of the skills I developed during the workshops included] documenting reactions, reaction mixes, tables of reagents, etc. interpreting… using controls, positive, negative controls on tests - so you know what you did wrong." - Reflections of a Biohacker on the workshops

Result: Analytical Digest of the Public Biobrick

Five colonies were picked from the ligation plates. After inoculation in LB + chloramphenicol overnight, these were minipreped and analytical digest was performed. All samples in lanes labelled one were digested once with Eco RI and samples labelled two are digested with EcoRI and Pst I. Colonies C and D clearly have the antifreeze insert. Lanes C2 and D2 show bands around 2000bp for the CMP plasmid backbone and around 400bp for the 435bp antifreeze region amplified by the primers. Therefore we have the biobrick!

Safety and regulations

To comply with UK/EU regulations on GMOs, we conducted transformation of our ligated plasmid into W3110 E.coli cells at UCL.

Making Competent Cells

Legality of Making Competent cell at London Hackspace Document

We attempted to make E. coli W3110 cells chemically competent at Hackspace. After long discussion and checking with relevant documents, we concluded that making these cells does not constitute genetic modification and therefore there is no legal restrictions on the creation of this competent strain.

We discussed health and safety issues at hackspace prior to starting any practical wetlab activities. It was important to hear what safety guidelines are normally followed at the hackspace and also brainstorm why it is important to follow these guidlines in the laboratory. Particularly, we focused on the potential risks of the reagents that are going to be used.