Encapsulation of Bacterial Ecosystems
Helena Shomar and Stephen Fortune
(See below for practical guide)
This practical session featured a novel biotechnological technique for encapsulation and immobilization of cellular organisms, using sodium alginate to form beads which would hold cultures of E. coli bacteria. The nature of the materials used also allows these enclosed environments to interact with each other.
The protocol offers confinement and immobilisation of living entities within man-made semi-permeable structures, the advantage over other methods being that things can enter and leave the membrane. One of many aims of this process is to organise the biological systems, controlling the in flows and out flows. In short – it’s like working with numerous nano petri dishes, and can be applied to drug cell delivery, bio nano-reactors, directed evolution and screening genetic libraries
The workshop also encouraged theoretical considerations of the concept of encapsulation within the biosciences alongside the coupling of organism and environment. Owing to the incubation/waiting periods within the protocol, time was available to engage with these themes and other related narratives and discussions pertinent to the practical and conceptual arguments of the session.
Following a presentation which introduced us to the two protocols “shine a light” (using a GM glow in the dark strain) and “lethal encounter” (a mixture of GM and wild type) we proceeded to suspend the bacterial culture in the alginate solution and create the beads by syringing it, painstakingly drop-by-drop into a calcium solution. Each bead is a micro-ecosystem – not membranous with liquid inside, but more like a gel, a matrix. The beads are then placed into the various nutrient solutions – some of which containing inducers and some antibiotics. The reactions of the combinations of the wild type and the GM will vary depending on which media they are cultured in.
The beads – which are now nano petri dish ecosystems – are left in this aqueous phase with the nutrients for some 30 minutes to allow take up of the media. After this they are subjected to the hydrophobic phase which pretty much seals the bead against further interactions with the outside world. When this stage is complete the beads are poured into petri dishes and taken away for incubation.
What is predicted to occur is this:
Shine a Light – the beads that will have physical contact will fuse with each other and the inducer will diffuse within both beads. The cells that were initially grown without the inducer in bead A will also become fluorescent thanks to the interaction/exchange with the other bead B.
Lethal Encounter – the beads that will have physical contact will fuse with each other = the antibiotic will diffuse within the bead carrying the wild type strain = the ‘natural’ E.coli will die due to the interaction with the environment of the GMO strain.
Results will be revealed tomorrow afternoon…
Total Time Requirements: 2 hours in the morning, 30 minutes in evening
This workshop needs to be staged first thing in the morning in order to provide time for growth of microbial colonies. An additional ‘coda’ period of up to 30 minutes at the end of the working day is requisite for participants to visualise the outcomes of their workshop labour.
- 15 mins suspending culture in alginate, and making beads by plungering them into the CaCl2 suspension
- 10 mins waiting for the calcium chloride (CaCl2) bath to take effect
- 30 mins incubation
- 15 mins separating and sorting some beads, putting them in the oil
- Approx 4-5h to overnight max
- Sterile 2% alginate solution (alginate solution can be filter sterilized, and autoclaved. Note: it has been reported that bead stability is reduced when the autoclave is used for sterilization.)
- Sterile 100mM Calcium chloride solution (CaCl2) - also very common, solution can be autoclaved or filtered for sterilization
- 20 mL syringes
- Glass beakers or container for recovery of the beads + magnetic shaker
- E.coli transformed with plasmid GFP (under inducible promoter) + antibiotic resistance
- Growth medium to determine, such as a sterile glucose solution
- Small plastic strainers strainers or sleeves to recover the beads
- (Laboratory) magnifying glass or microscope
- Transilluminator for observing fluorescence
- Chemicals: antibiotic corresponding to the plasmid resistance, inducer
Participants in groups prepare one of the two protocols
Case 1: A GMO strain that behaves differently depending on the environment
→ E.coli carrying GFP inducible plasmid. These strain is encapsulated then the batch is separated in different containers:
- A: 1 with growth medium (nothing happens)
- B: 1 with growth medium + inducer, which will make the cells green fluorescent (visible via magnification). Beads suspended in this medium will therefore contain the inducer
After 30 min of incubation in this medium, a fraction of both batches is recovered and mixed within mineral oil = exchange with the environment is disrupted. Of the beads suspended in mineral oil, those that will have physical contact will fuse with each other = the inducer will diffuse within both beads = the cells that were initially grown without the inducer in bead A will also become fluorescent thanks to the interaction / exchange with the other bead B.
Case 2: A wild-type strain and a GMO strain are separately encapsulated
→ We will use WT E.coli and E.coli carrying a plasmid with antibiotic resistance
- A: WT E.coli is grown in growth medium
- B: Antibiotic resistant strain in growth medium + antibiotic
After 30 min of incubation in this medium, a fraction of both batches is recovered and mixed within mineral oil = exchange with the environment is disrupted. The beads that will have physical contact will fuse with each other = the antibiotic will diffuse within the bead carrying the wt strain = the ‘natural’ E.coli will die due to the interaction with the environment of the GMO strain.