Biohack workshop, Design Interactions, RCA January 2014

Ira Ekaterina Back3
Genetically-modified E.coli cells: designed and produced by Design Interactions students at RCA
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
Bacterial transformation
Bacterial transformation (Ira, Jei & Tim)

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.

npuc18_19
DNA Plasmid ‘Circuit’ (image source http://dwb4.unl.edu/)

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.

Pipette close
Micro-pipetting: Naama & Rachel
biobricks
Genetic components (& reagents) ready for assembly

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

Beads
Adding micro-beads to DNA: Sam, Tim & Frank

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.

Small illustration
Yi-Wen, Rod, Naama & Anna
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.

transformation2
Bacterial transformation (Ira, Jei & Tim): One of the tactics to incorporate/sneak foreign DNA into bacteria is to create holes across cell membrane to allow gene absorption. To make such holes, cells must be stressed in some way, such as shocking with acute temperature changes in the presence of DNA circuit. Here the cells were kept ice cold and then subjected to 42C water bath (using an improvised mug) for 45 seconds. A second or two out, transformation is unlikely to work. [Photo credit: Sam Conran]
spreading
Anna spreading transformed cells onto fresh agar plate

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):

Ira Ekaterina Front1
Incubated cells ready for unwrapping
Ira Ekaterina Back2
Genetically-modified bacteria with our circuit containing an antibiotics resistance and LacZ gene (to produce blue compound). Since the plate contained antibiotics and X-gal (for LacZ gene to work on), the circuit assembly and transformation appear to have worked. Congratulations Ira and Ekaterina!
Ira Ekaterina Back6
As cells are spread onto the plate prior to incubation, a concentration gradient is formed across the plate where initial contact of the spreader creates highest cell concentration with the least being the last place of contact, which is often the edges of the plate. It may be that cell concentration prior to plating could have been adjusted for more even growth and an overall visually more satisfying result.
Sam Tim Frank Front2
Transformation and modification also seem to have worked for Sam, Tim and Frank. Apologies for other groups which did not produce any visible colonies: There is a suspicion that the earlier (morning) group had the short straw: The ‘blank’ cells for transformation may not have grown enough to provide sufficient cell population for effective transformation. A couple of hours of extra growth for the afternoon group seemed to have made a lot of difference – which perhaps highlights rapid nature of bacterial growth.
Sam Tim Frank Front3
Strong growth observed also towards the edge of the plate.
James Jei Front2
James and Jei’s plate was an interesting one: They have produced white colonies (not blue). This could be down to many different reasons but this may be due to partial modification. I.e. Rather than acquiring both antibiotics resistance and the LacZ gene (essential for blue colour), the DNA assembly may (or may not) have resulted a genetic ‘short circuit’ where only antibiotics resistance gene was included.
pUC19 Control
Positive control: Ready-made full circuit transformed and grown
Ira Ekaterina Clone
Any single bacterial colony can be transferred to a fresh plate and grown further, or cloned. Here are Ira and Ekaterina’s clones.
Sam Tim Frank Clone
Sam, Tim and Frank’s clones
James Jei clone
James and Jei’s clones
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/