Difference between revisions of "Part:BBa K3747606:Design"

Latest revision as of 21:41, 21 October 2021

Denitrification BAC

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].

Figure 1. Denitrification BAC carrying the whole denitrification machinery from Pseudomonas stutzeri.

In-depth design

From 5' to 3', the 32 kb denitrification casette consists of:

1. lox66(BBa_K3747604), synthetic variant of loxP, added to facilitate Cre-Lox-mediated genomic insertion. Sequenced retrieved from [1].

2. Gentamycin resistance(BBa_K3910008), to select for successful transformation of the BAC

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(BBa_K3747602) with at the 5’ end a T7 promoter: BBa_K3633015,a RBS: (BBa_J34801).

Nor is the nitric oxide reductase, which converts nitric oxide (NO) into nitrous oxide (N₂^-O). Cytochromes and genes for heme d₁ synthesis are present in the native operon. Without these co-factors, Nir would not have been active. Nor takes care of the second step of denitrification and was therefore added as the third 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. ALthough the T7 phage's genome is 40kb long, The T7 polymerase is added to make sure the last two enzymes (Nor, Nos) are expressed.

6. The nitrous oxide reductase (Nos) operon from P. stutzeri(BBa_K3747603) with at the 5’ end a RBS: (BBa_J34801).

Nos is the nitrous oxide reductase, which converts into nitrous oxide (N₂^-O) into dinitrogen gas (N₂). Nos takes care of the last step of denitrification and was therefore added as the last 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.

7. lox71/2m(BBa_K3747605), synthetic variant of loxP, added to facilitate Cre-Lox-mediated genomic insertion. Sequence retrieved from [1].

Source

Pseudomonas stutzeri JM300

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.