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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 (BBa_J34801) 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 integrate 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 lox71 and lox66 2m (retrieved from [1]) and a T7 terminator (Figure 1). To facilitate integration, we flanked the denitrification machinery with gentamycin resistance cassette with lox66 and lox2m/71 [1]. 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.

Figure 6. Landing pad cargo for the integrative pGNW vector. With this construct, from 5’ to 3’, the T7 terminator, lox66, Cre recombinase, lox71 and the T7 promoter are integrated. This is because H1-cusF and H2-cusF correspond to regions at the 3’ and 5’ end of the PP_5388 site respectively.

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 [2]. 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.

Figure 6. IPTG inducible T7pol cargo for the integrative pGNW vector. With this construct, from 5’ to 3’, the lacI promoter, lacI, the lac operator, T7 polymerase and &lambda₀ is integrated. This is because H1-glmS and H2-glmS correspond to regions at the 5’ and 3’ end of the attn7 site respectively.

Testing the casette

Within the timeframe of the iGEM competition, we were able to test NO₂^- and N₂O accumulation. 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. Curious about the results, check the experience page.

References

[1]Garcia-Morales, L., Ruiz, E., Gourgues, G., Rideau, F., Piñero-Lambea, C., Lluch-Senar, M., ... & Lartigue, C. (2020). A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae. ACS synthetic biology, 9(10), 2737-2748.

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