Date 10 January 2014 Lab Mechatronics room, Darwin Building, Royal College of Art, UK Participants 1st year students at Department of Design Interactions Risk Assessment Completed
Biohack workshop was held at the Royal College of Art, in conjunction with launch of its annual synthetic biology project for first year Design Interactions students. Core purpose of the workshop was threefold: To promote awareness and understanding of biotechnology, de-mystification of methodologies involved, and to enable designers to engage in molecular and genetic strategies. These were achieved predominantly through hands-on activities, involving full student participation involving use of several tools and protocols employed by modern research professionals.
Workshop provided a particular angle of approach to the project brief, which challenged students to ‘Re-design an Ecosystem’, by allowing them to think about a biological/living system from the bottom up, starting from the very basic unit of life, the DNA.
One of the activities included design and assembly of a genetic circuit (modified from above model) – consisting of several selected genes – that was transformed into a common, industry-standard E. coli K12 strain. Once inside a cell, the circuit allows host bacteria to express a functional LacZ gene (highlighted in grey block as per above map) which would turn the cells blue in the presence of a special growth medium containing X-gal.
Prior to the experiment, genetic circuit design was discussed, taking types of sequence for inclusion, desired functionalities of gene expression and the order of DNA assemblage into consideration. It was highlighted that following genes were essential (but not exclusive to) in making an efficient and visually tangible genetic modification in E. coli:
1. High copy number – to maximise gene expression
2. Antibiotic resistance – to positively select transformed cells
3. LacZ gene – responsible for producing an active enzyme which would produce blue compound when grown in X-gal-rich medium
4. Cap sequence – to literally ‘cap off’ the circuit and allowing circularisation and stabilisation of the plasmid
Circuit assembly began with adhesion of first genetic component to tiny metal structures called micro-beads. This formed a backbone of our circuit whereupon further biobricks could be added – or ‘ligated’ – onto the end of preceding DNA strand. Each ligation required an addition of a special enzyme called T4 DNA ligase (which has its origins from a bacteria-invading virus). Scale of such molecular process were too small to be observed by the naked eye, and required special tools such as micro-pipettes to allow effective transfer of tiny droplets of DNA-containing solutions from one reaction tube to the other.
Final circuit assembled:
AncXA-OriblaAB-LaczBA-CapAX
Complete DNA sequence available upon request
Once complete, circuits were then inserted into E. coli cells, using a technique known as ‘heat shock transformation’: This involved transfer of cells from ice-cold temperatures to a relatively warmer ‘bath’ (42C) at a measured timeframe. Cells were then returned to ice and fed nutrients to recover before being spread onto sterile agar plates containing antibiotics and X-gal for overnight incubation and growth.
Agar plates containing transformed cells were incubated overnight at 37C – which is an optimum temperature for E. coli growth. Following results were observed in some participants’ plates (after 12 hours of growth):
Special Thanks: Douglas Ridgway (Genomikon) for parts supply, Mike Alexander (RCA) for advice and risk assessment, Johanna Schmeer(assistance) and Design Interactions at RCA for hosting the workshop. Further info on health and safety implemented can be found at http://www.hse.gov.uk/
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