Kill Switches

Introduction

Synthetic Biology has a vast array of potential applications beyond the limits of a sterile laboratory; from the degradation of explosive residue left in soil to enhancing the As a result, it is necessary to implement control mechanisms over these synthetic organisms to ensure that they continue to function correctly and can remain contained to their intended environment.

What are they?

Kill switches are devices incorporated into synthetic organisms which offer a potential mechanism of regulating the life cycles of the organisms termed induced lethality. They are often designed such that they operate to terminate the organism when its intended function has been fulfilled.

Kill switches will often comprise an inducible promoter, for the purposes of laboratory testing this will likely be responsive to a simple common molecule which can be added to growth media, for example a sugar such as arabinose. However, this would ideally be responsive to an inducer molecule the concentration of which or even presence or absence in the cell is indicative of the progression or completion of the cell’s intended function.

Why we wanted one

As our synthetic E.coli are designed to degrade the residual explosives found in the ground at munition waste sites they would in theory be used actively in the soil. It would not be possible to simply release synthetic organisms into the environment without implementing essential biosafety mechanisms and it was therefore necessary for our project to include a kill switch.

Our initial thoughts/ideas

Looking into Epigenetic modifications and how these could influence gene expression over time.
Long term genetic stability of the population or individual parts – Biobrick stability
A device which could activate a lytic cycle of bacteriophage, their DNA incorporated into our bacteria
A method of interfering with quorum sensing e.g. the absence of TNT terminates QS and then a second signal becomes activated to kill the cells.
Using our synthetic Promoter (indirectly sensitive to TNT) to activate the kill switch device when the levels of TNT are low enough
RNA aptamers to TNT
Then having a timer so that it would continue to degrade the explosives even below the low triggering concentration
Timer using small cis/trans acting RNA molecules

Initial Research

It was decided that we would be investigating the retention of function of the kill switch over time. The notion that kill switches are inherently unstable was discussed as bacteria containing a device designed to kill it are at a significant competitive disadvantage compared to those without or those acquiring even a small mutation if it were enough to render the device functionally less active or completely inactive therefore allowing the cells to live.

In order to test the kill switch part over time, research was done into the E.coli long term evolution experiments http://en.wikipedia.org/wiki/E._coli_long-term_evolution_experiment in which the separate populations of E.coli are kept growing and each day 1% of the culture is transferred to the next container of minimal media. This set up would have been possible in our lab though may have required a lot of space in the shaking incubators and unfortunately there were issues with contamination.

Another source of inspiration for the work came from the Jove set up of multiplex chemostat arrays (http://www.jove.com/video/50262/design-and-use-of-multiplexed-chemostat-arrays). The idea that it would not be necessary to physically move cultures by hand was appealing although it proved difficult to source all of the necessary equipment.

It was decided instead to monitor the cells as they grow under different conditions in a small high throughput manner. Using a 96 well microplate and plate reader it was possible to obtain a lot of data about how growth is affected. The online registry was searched and many different parts used in previous kill switch designs were found including toxin-antitoxin systems for example, holin/antiholin or MazF/E in bacillus, there were also RNA exonucleases and mechanisms of inhibiting of DNA gyrase on the registry. After assessing multiple parts it was decided that part K628006 was to be selected for testing.

Part BBa_K628806 (http://parts.igem.org/Part:BBa_K628006) comprises a gene encoding an antimicrobial peptide under the control of a pBad arabinose inducible promoter. It was selected from a list of many potential kill switch parts as it had a simple mechanism of activation and results recorded on the parts registry showed successful killing over a time period of a few hours. It was decided that this part could be tested to determine whether this killing function was retained over a longer period of time.

Our Experiments

Overnight growth at different concentrations of arabinose (29/07/14)
6 hour monitored growth (30/07/14)

Used the 96 well microplate and plate reader to measure OD at regular intervals.
Used four different concentrations of arabinose

10 hour monitored growth (31/07/14)

96 well microplate
Included plain LB and Top10 controls
3 different concentrations of arabinose

Making plates containing different concentrations of arabinose and counting colonies – though these always produced a lawn of growth and couldn’t be counted.

Results and Conclusions from Experiments

That the part does not completely inhibit growth in the presence of the inducer molecule, arabinose as both the 6 hour and 10 hour experiments produced growth of transformed cells. However the shape of the growth curve produced is altered from typical sigmoidal or ‘S’ shape to something more linear with a peak in optical density before it appearing to begin a plateau or slight decline, though there is no data beyond the 10 hour limit.

Interestingly there seems to be no lag phase in the transformed culture and the population begins to grow straight away. This seems to suggest it was taken from an exponentially growing population. However all of the samples in this experiment were taken from overnight cultures which should have reached stationary phase. It could potentially be the case that what we are seeing is just the top of a sigmoidal curve. This would suggest that the population grows in a normal manner but just much more slowly than the untransformed cells.

When comparing the three graphs at different concentrations of arabinose, it can be seen that the peak in optical density is reached at progressively later times as the concentration of arabinose in the solution is increased. This difference in growth pattern may be due to the break down of arabinose or uptake by cells decreasing the concentration in the external environment therefore allowing growth when noramlly cells would have died.

This raises the question of stability and retention of function. If the cells are able to grow at high concentrations of the inducer molecule it may be indiciatve that the part is not functioning correctly and failing to kill the cells as intended.

Why we did the long term growth – why we deserve gold!

The kill switches aspect of the project contributes towards the teams efforts to meet the requirements for a gold medal in two ways. Firstly, through the characterisation of part: K628006 http://parts.igem.org/Part:BBa_K628006 from St Andrews in 2011. Secondly as it is an investigation into potential safety mechanisms that could be used in our project, highlighting potential problems that could be encountered in future.

Future Plans

If Exeter were to design and make our own kill switch for this project this year, it would be desirable to incorporate the synthetic promoter construct capable of indirectly sensing TNT. The absence of which would activate the killing function.

For Exeter 2014 our work on kill switches is only a small side part of the project though it would be interesting to investigate in more depth in future. Other teams have in the past looked at preparing synthetic biology for the world and whole iGEM projects could be built around this idea, for for example Imperial’s development of the geneguard system in line with DSTL’s biosafely initiative. http://pubs.acs.org/doi/abs/10.1021/sb500234s https://2011.igem.org/Team:Imperial_College_London/Project_Gene_Overview

Stop using antibiotics as markers – Engineered Auxotrophs
No reliance on kill switches
No genetic pollution – prevention of cell lysis
Modularity – plug-in parts
Engineering dependency of plasmids on their host strains
Making plasmids disadvantageous for the hosts
Conditional origins of replication

It would be desirable to look at the long term function of these sorts of devices and use this information to make a much more informed decision on how trustworthy these parts are. If synthetic biology is to be applied to the real world we must be able to correctly predict whether, and if so how, these organisms will change over time.

Exeter | ERASE

Team:Exeter/Kill Switches

From 2014.igem.org