Team:Bristol/Parts

The NOx gases NO and NO₂ dissolve to form nitrite (NO₂^-) and nitrate (NO₃^-) in solution. Nitrate can be converted into N₂ by a process called denitrification. N₂ can then be converted to ammonia by nitrogen fixation, which occurs in the soil and is carried out by nitrogen fixing bacteria. However, this process requires many different enzymes, some of which are not expressed in E. coli. Instead, we are using a process called dissimilatory nitrate reduction to ammonium (DNRA), in which nitrate is reduced to ammonium directly. In E. coli this process requires 2 enzymes: NapA and NrfA.

The Denitrification Pathway

Operon Key

Nrf

The Nrf operon encodes the E. coli cytochrome c nitrite reductase, which catalyses the 6 electron reduction of nitrite (NO₂^-) to ammonia (NH₃) [1]. NrfA encodes the catalytic NrfA homodimer, which forms a heterotetrameric complex with the homodimeric NrfB gene product which facilitates electron transfer from the membrane-bound NrfCD complex. We intended to provide all components of the operon within one part, to ensure full activity of NrfA.

The operon is under the control of an IPTG inducible tac promoter (with lac operator). Our designed part includes the LacI gene, along with its promoter and RBS.

The Nrf Operon

Nap

The Nap operon encodes the periplasmic nitrate reductase, which catalyses the single electron reduction of nitrate (NO₃^-) to nitrite (NO₂^-) [2]. We utilise Nap to maximise the amount of NO₂^- that can feed into Nrf, as NO₃^- is not an Nrf substrate. The membrane bound component NapC accepts electrons from the quinone pool, and transfers these to the NapAB catalytic core. NapG and NapH are proposed to act together as a quinone dehydrogenase [2]. NapD and NapF are also essential.

The Nap operon is also controlled by the tac promoter and lac operator (IPTG inducible). When in conjunction with Nrf, this allows both operons to be induced simultaneously. This part does not include the LacI gene, or its promoter.

The wild type Nap operon also contains the c-type cytochrome maturation (Ccm) enzymes. However we removed these, as they will be provided on a separate plasmid.

The Nap Operon

Composite Part

We ultimately planned to submit our Nrf and Nap parts as a composite part. This would allow conversion of E. coli to an efficient NOx conversion bioreactor with one simple transformation. The operons are separated by a double terminator to ensure individual expression.

Submitted Part

Unfortunately, we were not able to assemble any of these full constructs. In order to still contribute to the iGEM registry, we edited our Nrf2 fragment so isolate the whole NrfB coding sequence (see our Experiments page), and submitted this as an individual part (part BBa_K2293000). NrfB is essential for the expression of functional NrfA, so addition of the coding region to the iGEM registry will allow future teams to fully express NrfA, using our NrfB part with the already existing NrfA sequences in the registry.

References

1. Mechanism of the six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase. Einsle O, Messerschmidt A, Huber R, Kroneck P, and Neese F. Journal of the American Chemical Society 2002 vol: 124 (39) pp: 11737-11745

2. Nitrate and periplasmic nitrate reductases. Sparacino-Watkins C, Stolz J, Basu P. Chemical Society reviews 2014 vol: 43 (2) pp: 676-706