Team:USTC/Kill Switch





Light Activated Kill Switch

Our bacteria is designed to be used in anaerobic tower at waste water treatment plant. (Click here to learn about the details) To prevent our bacteria from escaping to environment, we decided to introduce a light activated kill switch. We found one from Team Unesp Brazil 2018.

Mechanism

Photoreceptor VVD is used as the “switch”[2]. The engineered variants are named Magnets (pMag and nMag). Positive (BBa_K2660009) and negative (BBa_K2660008) magnets (pMag and nMag) dimerize in response to blue light through electrostatic interactions (Fig. 1).

Cas9 is used as the “killer”. Guided by gRNA, Cas9 will cut down the target sequences, causing cell death. N-terminal and C-terminal domain of Cas9 are separately linked to magnets by a 10 aa linker (BBa_K105012). Therefore, Cas9 is activated only when pMag and nMag dimerize under blue light.

Design of gRNA

The gRNA consists of target, PAM, and scaffold sequences. To ensure cell death when necessary, we targeted essential genes like RNA polymerase B (rpoB), DNA polymerase III and DNA ligase. We designed the target and PAM sequences with the help of an online tool[3]. Two versions of gRNA were constructed, with the highest or second highest estimated on target rate (Table 1). And the sequence of scaffold was found from a handbook.

Table 1 Estimated on target rate
# Ver 1 Ver 2
rpoB 1 1
DNA polymerase III 0.96 0.94
DNA ligase 0.88 0.88
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Figure 6. Sequences of gRNA

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Figure 7. Light Activated Kill Switch

Reference

[1] https://2018.igem.org/Team:Unesp_Brazil/Design

[2] KAWANO, F. et al. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. v.6 n.6256, 2015. [3]https://sg.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE