Part:BBa_K3747606:Design
Denitrification BAC
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Illegal EcoRI site found at 1957
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Design Notes
Given that the denitrification machinery + antibiotic resistance is about 32kb, a BAC was designed. This was done in a way that it could be assembled in yeast. Therefore, a centromere (CEN), an autonomous replicating (ARS), and Leucine biosynthesis (LEU) were included in the design [1]. Leu was added because the yeast strain that would have been used is auxotrophic for leucine. Moreover, we used the PCC1fos backbonewhich is a low copy number plasmid. The rationale for this is that a large, high copy number plasmid >30kb can excessively burden bacteria and are prone to mutate [2].
In-depth design
From 5' to 3', the 32 kb denitrification casette consists of:
1. lox66, synthetic variant of loxP, added to facilitate Cre-Lox-mediated genomic insertion.
2. Gentamycin resistance, to select for successful transformation of the BAC (BBa_K3910008)
3. The periplasmic nitrate reductase (Nap) operon from P. stutzeri (BBa_K3747600) with at the 5’ end a ribosome binding site (RBS): (BBa_J34801). Nap is the dissimilatory nitrate reductase, which converts nitrate (NO3-) into nitrite (NO2-). This is the first step of denitrification, and therefore added as the first denitrification enzyme in the BAC. The RBS is added to ensure transcription of the first gene of the operon, as during PCR amplification the native RBS could have been lost.
4. The nitrite reductase (Nir) operon from P. stutzeri(BBa_K3747601) with at the 5’ end a ribosome binding site (RBS): (BBa_J34801).Nir is the nitrate reductase, which converts nitrite (NO2-) into nitric oxide (NO). Cytochromes and genes for heme d1 synthesis are present in the native operon. Without these co-factors, Nir would not have been active. Nir takes care of the second step of denitrification and was therefore added as the second enzyme in the BAC. The RBS is added to ensure transcription of the first gene of the operon, as during PCR amplification the native RBS could have been lost.
5. The nitric oxide reductase (Nor) operon from P. stutzeri with at the 5’ end a T7 promoter: BBa_K3633015 RBS: BBa_J34801
6. The nitrous oxide reductase (Nos) operon from P. stutzeri with at the 5’ end a RBS: BBa_J34801
7.
Source
P. stutzeri JM300 (majority)
References
[1] N. Kouprina and V. Larionov, “Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes,” FEMS Microbiol. Rev., vol. 27, no. 5, pp. 629–649, Dec. 2003, doi: 10.1016/S0168-6445(03)00070-6. [2] G. DG, Y. L, C. RY, V. JC, H. CA, and S. HO, “Enzymatic assembly of DNA molecules up to several hundred kilobases,” Nat. Methods, vol. 6, no. 5, pp. 343–345, 2009, doi: 10.1038/NMETH.1318.