Difference between revisions of "Part:BBa K3747606"

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Denitrification BAC

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Sequence and Features

We created a bacterial artificial chromosome (BAC) carrying all four denitrification operons, Nap, Nir, Nor, Nos from P. stutzeri JM300. To guarantee complete transcription of the 32 kb cargo, a T7 promoter ( was added in the middle, between the Nir and Nor operons. Moreover, at the 5’ end of each operon, an RBS (BBa_J34801) was added. High copy number plasmids are normally employed to continuously propagate the system. However, high copy number plasmids containing a large cargo can excessively burden the bacteria and are prone to mutate. Therefore, we integrated the complete cargo in the genome of P. putida EM42.

Integration Approach

To transport the complete denitrification machinery, we prepared a ‘landing pad’ in P. putida EM42. The landing pad comprises a T7 promoter that controls the expression of Cre recombinase flanked by two lox sites. To facilitate integration, we flanked the denitrification machinery with gentamycin resistance cassette with lox66 and lox2m/71. After successful conjugation of the BAC into P. putida EM42 ∆nasT, transiently expressed Cre recombinase recognized the lox sites and subsequently integrated the denitrification machinery.

Expression of the casette

Given that we placed the denitrification pathway under the control of 2 T7 promoters, we needed to integrate a T7 polymerase (T7pol). This polymerase specifically transcribes DNA only downstream of a T7 promoter, synthesizes RNA at a high rate. Additionally, the T7pol ignores terminators which increases the likelihood that the whole pathway is transcribed [1]. Moreover, we made T7-polymerase expression IPTG inducible. This was done to prevent constitutive expression, and with that the associated metabolic burden of the denitrification cassette. The final strain was called P. putida :SD, SD standing for Synthetic Denitrification.

Testing the casette

Within the timeframe of the iGEM competition, we were able to test NO₂^- and N₂O accumulation. For NO₂^- accumulation specifically, the strains were grown with NO₃^- for 24 hours. Furthermore, we tested the effect of IPTG on nitrogen dynamics, the strains were grown with with IPTG (:SD+) and without IPTG (:SD). For the :SD+ condition, the pathway is transcribed to a higher extend. Compared to the control, more NO₂^- accumulates for :SD and :SD+, suggesting that the NO₃^- reductase works. However, when compared with the Nap plasmid, less NO₂^- accumulates. This could imply two things: (1) nitrate reduction is not as optimal as for the plasmid or (2) the coupled NO₂^- reductase works too. The fact that the :SD with IPTG accumulates less NO₂^- compared to the :SD suggests that more NO₂^- is reduced. This could be explained by higher expression rates downstream of the pathway.

References

[1]T. S, “Expression using the T7 RNA polymerase/promoter system,” Curr. Protoc. Mol. Biol., vol. Chapter 16, no. 1, Jul. 2001.