Part:BBa_K4849003:Design
NucA nuclease from Anabaena sp. PCC 7120
Design and Experimental confirmation
Our intention was to use the NucA nuclease as a toxin in the kill switch, thus to optimize it for this purpose we made a couple of modifications to the gene. First, in order to achieve intracellular localization of the nuclease and to make sure it cuts cellular DNA upon kill switch induction, we shortened the coding sequence of nucA by 69 nucleotides to remove the signal peptide mediating the export of nuclease to the periplasm (Muro-Pastor et al., 1992). Second, it was previously observed that for the efficient cloning of the nuclease-based kill switch degradation tags had to be introduced to curtail the cellular half-life of the nuclease (Čelešnik et al., 2016). We added the T1 degradation tag (RPAANDENYAAAV) (Huang et al., 2010) to the C-terminal of the NucA nuclease (Huang et al., 2010). Finally, we synthesized the modified nucA sequence with added overhangs suited for CyanoGate (Vasudevan et al., 2019) assembly, and ligated it into a Level 0 acceptor vector for CDS1(Engler et al., 2014) by BbsI assembly (Gale et al., 2019). The assembled Lv0-NucA construct was transformed into competent Escherichia coli TOP10 cells. Colony PCR was used to screen for colonies with the correct insert size. The expected amplicon size for Lv0-NucA was 1120 bp. All five screened colonies gave the correct band (Figure 2).
Figure 2. Colony PCR of Level 0 constructs.
One of the colonies with the correct cPCR band for NucA was screened by restriction digestion with BsaI (cuts out the insert) and PvuI (linearizes the plasmid), and the digested DNA was analysed by gel electrophoresis. The DNA bands for the digested Lv0-NucA construct were of approx. expected sizes, 2247 bp and 796 bp (Figure 3).
Figure 3. Restriction digestion of Level 0 constructs.
We also sent the Lv0-NucA construct to Edinburgh Genome Foundry for sequencing by Oxford Nanopore technology and analysis. The sequencing analysis report showed that most reads of the Lv0-NucA construct (barcode05/ lv0_NucA) were of the expected size. The ‘coverage plot’ showed that the entire plasmid was observed. From comparison with reference sequence (Figure 4), some ‘True’ mutations were detected in the backbone of the construct, but not in the insert region.
Figure 4. The table lists mutations detected in the Lv0-NucA construct. Those highlighted in red are ‘True’ mutations, whereas those not highlighted are sequences known to be challenging to accurately sequence with nanopores (usually secondary structures / repetitive sequences). In this table, DP is how many reads cover this part of the sequence, RO is how many of those reads contain the expected sequence, AO is how many contain an alternate allele.
References
Čelešnik, H., Tanšek, A., Tahirović, A., Vižintin, A., Mustar, J., Vidmar, V. and Dolinar, M., 2016. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biology Open, 5(4), pp.519-528.
Engler, C., Youles, M., Gruetzner, R., Ehnert, T.M., Werner, S., Jones, J.D., Patron, N.J. and Marillonnet, S., 2014. A golden gate modular cloning toolbox for plants. ACS synthetic biology, 3(11), pp.839-843.
Gale, G.A., Osorio, A.A.S., Puzorjov, A., Wang, B. and McCormick, A.J., 2019. Genetic modification of cyanobacteria by conjugation using the CyanoGate modular cloning toolkit. JoVE (Journal of Visualized Experiments), (152), p.e60451.
Huang, H.H., Camsund, D., Lindblad, P. and Heidorn, T., 2010. Design and characterization of molecular tools for a synthetic biology approach towards developing cyanobacterial biotechnology. Nucleic acids research, 38(8), pp.2577-2593.
Muro‐Pastor, A.M., Flores, E., Herrero, A. and Wolk, C.P., 1992. Identification, genetic analysis and characterization of a sugar‐non‐specific nuclease from the cyanobacterium Anabaena sp. PCC 7120. Molecular microbiology, 6(20), pp.3021-3030.
Vasudevan, R., Gale, G.A., Schiavon, A.A., Puzorjov, A., Malin, J., Gillespie, M.D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C.J. and Lea-Smith, D.J., 2019. CyanoGate: a modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant Physiology, 180(1), pp.39-55.
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