Part:BBa_K2109111
mazF kill switch improvement
This is a composite killswitch based upon BBa_K1405008 submitted by the BNU-China 2014 team. MazF is an endoribonuclease which initates a cell death pathway which can be co-opted in a biocontainment strategy. In essence, the killswitch proposed by BNU-China's 2014 allows for programmed cell death upon induction with IPTG. Use of this killswitch was prompted by the collaboration between the Lethbridge and Marburg 2016 teams. Throughout this collavoration, the Marburg team contributed mathematical modelling information regarding the evolutionary stability of killswitches while the Lethbridge team experimentally tested their hypotheses.
This is an improvement upon the previous MazF killswitch part (BBa_K1405008). When the previous part was obtained it did not performa as expected. Upon sequence analysis it was noted that BBa_K1405008 lacked any ribosome binding sites for protein production (including one for MazF toxin production). To this end, ribosome binding sites were inserted for production of MazF. Additionally, due to the leakiness of the previous regulation by IPTG, this part was placed under control of a tighter regulatory system (pBAD promoter under control of arabinose induction.
The Lethbridge 2016 team then wished to assess the escape frequency of the new killswitch part. This is an important consideration for a biocontainment project of this nature. A collaboration was formed with the Marburg 2016 team in which the MazF part was mathematically modelled and the evolution of killswitches were assessed. Certainly, a killswitch part such as this is under constant evolutionary pressure. The predictions made by Marburg 2016 and their modelling expertise were assessed by the Lethbridge 2016 team. The evolutionary stability of the killswitch part was assessed by subjecting E. coli cells harbouring the killswitch part to ultraviolet light. Equal numbers of cells were subjected to ultraviolet light and plated in the presence and absence of the inducer molecule. The expected result in the presence of arabinose is non-viable cells, which is what was observed. After subjecting cells to UV light and plating, several cells escaped the killswitch part, indicating that mutations had possibly been accrued to make the killswitch ineffective. The CFUs which escaped the killswitch will be sequenced to determine where mutations occurred and how this might impact the stability of killswitches over time. This will be a part of the ongoing collaboration with the Marburg 2016 team.
Figure 1. Plates containing the MazF killswitch (K2109111) before and after UV treatment.
EXPERIMENTAL PROTOCOL:
500,000 microjoules of UV light was used to irradiate 200 microlitres of E. coli cells (OD600=0.1) containing an inducible plasmid expressing MazF. Cells were then plated on LB+agar plates containing antibiotic and inducer and grown for 16 hours at 37 degrees Celsius. The number of CFUs obtained after UV irradiation was compared to prior to irradiation and an escape frequency was obtained using this method. Clones which escape the killswitch will be sequenced to examine possible mutations allowing for their escape. Optimization of the amount of UV delivered as well as replication of these experiments are required, however it is a valuable step forward in assessing the evolution of killswitches.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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