Part:BBa_K4849004:Design
NuiA antitoxin from Anabaena sp. PCC 7120
Design and Experimental confirmation
Our intention was to use the NuiA as an inhibitor in the NucA-based kill switch. We synthesized the modified nuiA 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-NuiA 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-NuiA was 733 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 409 bp (Figure 3).
Figure 3. Restriction digestion of Level 0 constructs.
We also sent the Lv0-NuiA construct to Edinburgh Genome Foundry for sequencing by Oxford Nanopore technology and analysis. The sequencing analysis report showed that most reads of the Lv0-NuiA construct (barcode01/ lv0_NuiA) were of the expected size or a bit larger (due to the addition of the barcode sequence). 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-NuiA 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
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.
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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