Part:BBa_K5111009
ABC Transporter, copper chaperone and TAT exporter
In this insert, we have added the CDS of the other nos cluster genes – NosDFY and NosL – and the tatE protein. NosDFY codes for an ABC transporter code for an ABC transporter which provides sulphur atoms to the CuZ centre in N2OR (Bennett et al., 2020). nosL codes for a lipoprotein/copper chaperone which binds to copper and is required for the assembly of the CuZ centre (Bennett et al., 2019). It may also play a role in the maturation and activation of N2OR. tatE supports the Tat-dependent export of apo-NosZ (Bennett et al., 2019). Together with our first NosR system (BBa_K5111008), this insert can produce the nitrous oxide breaking protein N2OR. The genome for these proteins were derived directly from P. stutzeri (Clade I) ZoBell ATCC 14405 meaning we have kept the natural RBSs present in between the open reading frames of the proteins. P. stutzeri has also shown to have the greatest enzymatic activity of N2OR under anaerobic and aerobic conditions (Yoon et al., 2016). Thus, we believe these two nos inserts would perform well in both scenarios.
Sources Bennett, S.P. et al. (2020) 'nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase,' Microbiology, 166(10), pp. 909–917. https://doi.org/10.1099/mic.0.000955.
Bennett, S.P., Soriano-Laguna, M.J., Bradley, J.M., Svistunenko, D.A., Richardson, D.J., Gates, A.J. and Le, N.E. (2019). NosL is a dedicated copper chaperone for assembly of the CuZcenter of nitrous oxide reductase. Chemical Science, 10(19), pp.4985–4993. doi:https://doi.org/10.1039/c9sc01053j.
Yoon, S., Nissen, S., Park, D., Sanford, R.A. and Löffler, F.E. (2016). Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ. Applied and Environmental Microbiology, 82(13), pp.3793–3800. doi:https://doi.org/10.1128/aem.00409-16.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2237
Illegal BglII site found at 3025
Illegal BamHI site found at 1850 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 207
Illegal NgoMIV site found at 402
Illegal NgoMIV site found at 1006
Illegal NgoMIV site found at 2100
Illegal NgoMIV site found at 2819
Illegal NgoMIV site found at 3037
Illegal NgoMIV site found at 3180
Illegal AgeI site found at 3260 - 1000COMPATIBLE WITH RFC[1000]
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