Team:UCL/Science/Proto
From 2014.igem.org
In this section we will look at our project and synthetic biology from a very different perspective. As students of the emerging field of synthetic biology we feel very much drawn towards the science and technology that makes our project possible. However, as scientists in the making we are part of a society which means that this project also provides us with the opportunity to reflect on how our work affects and is affected by the social and ecological environment in which we work. We therefore have to imagine how our current and future societies would go about dealing with the implications of synthetic biology. This kind of reflection has been termed by the American sociologist C. Wright Mills as sociological imagination in order to describe our awareness of how individual experience and the wider society relate to one another (Mills 1959).
Here we will use our sociological imagination to look at how our project can help to conceive a sustainable governance model for synthetic biology. The Goodbye AzoDye Project is instrumental in achieving this because it enables us to explore and examine the dual nature of the technology as synthetic biology promises to be highly beneficial to society while at the same time creating increasing uncertainty in terms of incalculable risks and issues of biosecurity. There are signs that the community of synthetic biologists is prone to be confronted with a potential public controversy revolving around the environmental hazards that their dual-use technology can be perceived to bring to society. At the same time, however, by taking part in the Environment Track of the Giant Jamboree, UCL iGEM 2014 is also engaged with the idea of solving an ecological problem. Hence, a paradox emerges within the team in which discourses of environmental decline - in relation to the misuse of synthetic biology and the problem of azo dye effluents - are forced to coexist with discourses of environmental innovation to solve these problems.
Considering the complex and novel nature of scientific practices in synthetic biology there is a need to look at adapted forms of governance that deal with processes of innovation in a reflexive manner. This is seen as necessary in order to devise policies that can accommodate a sustainable development of the emerging technology within society. Considering the environmental risks to which they are ascribed, policy frameworks ought to engender effective governance that seeks to foster good science, not to hamper it. It also recognises that good science goes hand in hand with open, clear, transparent regulation to ensure both trust and accountability. Another prominent feature of synthetic biology is its ‘cross-borderness’, in addition to the embedded scientific uncertainty. It simultaneously crosses the borders of scientific disciplines, industrial sectors, and geopolitical areas. Considering the transboundary and uncertain nature of this emerging technology it might be interesting to look at how policies are being developed within the framework of transnational governance. Some views support the idea that synthetic biology policies should not only be regulated from a top down perspective through governments, but that non-governmental stakeholders and organisations should be able to engage in self-regulation. The transboundary – and transnational nature of synthetic biology practices makes it pertinent to examine biosecurity and sustainable innovation discourses at the level of transnational governance structures such as iGEM. The latter holds a series of promising characteristics with regard to innovative regulatory frameworks.
Key words: dual-use governance - environmental discourse - ecological modernisation - risk society - UCL iGEM 2014 - sustainability - uncertainty - reflexive modernisation
Creating Competent Cells
LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)
Procedure
1. Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC
2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.
Shake @ 37oC for 1.5-3 hours.
Or
1. Inoculate a single colony into 25ml LB in a 250ml bottle in the morning
2. Shake @ 37oC for 4-6 hours.
Then…
3. Put the cells on ice for 10mins (keep cold from now on).
4. Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.
5. Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).
6. Incubate on ice x 20 minutes
7. Centrifuge as in 2.
8. Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.
9. Dispense in microtubes (300ųl/tube). Freeze at -80oC.
Transformation of competent cells
Competent Cells, Plasmid DNA, Antibiotic Plates
Procedure
1. T haw competent cells on ice
2. 50uL cells enough for 1 transformation
3. Add 1ug of DNA to 50uL competent cells
If biobrick from distribution, resuspend DNA well in 10uL ddH20
4. Add 1uL biobrick DNA to 50uL competent cells
5. Add 1uL RFP control to 50uL competent cells for your control transformation
6. Flick by hand or pipette up and down gently
7. Place cells on ice for 30 minutes
8. Place cells in water bath at 42oC for 40 seconds
9. Place cells on ice for 2 minutes
10. Add 0.5mL of LB media and place in incubator for a maximum of 2 hours (37oC/250rpm)42oC (200 µl SOC media can be used to improve transformation efficiency)42oC 11. Label two petri dishes with LB agar and the appropriate antibiotics(s) with the part number, plasmid backbone and antibiotic resistance
12. Plate 50 µl and 500 µl of the transformation onto the dishes, and spread.
13. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up.
If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria
You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.