Difference between revisions of "Part:BBa K4259006"

Latest revision as of 10:24, 11 October 2022

napA from E. coli

NapA is part of the periplasmic nitrate reductase (Nap), one of the three Nitrate Reductases in E. coli.¹

Usage and Biology

Binanox aimed to synthesize bimetallic nanoparticles with a silver core and golden spikes by overexpressing certain genes in E. coli by using a cell-free system. It has been shown that NADH and NADH-dependent enzymes are important factors in the biosynthesis of metal nanoparticles, especially the presence of nitrate reductase might be an integral part in the synthesis of silver nanoparticles. Nitrate is converted to nitrite during the reduction process and an electron will be transferred to the silver ions. The result is that silver ions are converted to elemental silver.¹ NapA was specifically chosen as it has a high affinity for nitrate.² In previous studies it was seen that it has emerged as a strong candidate for the production of silver nanoparticles (AgNP) in bacteria, especially in a nitrate-rich medium. Nitrate-rich media are known inducers of nitrate reductase. It was shown that AgNP absorbance was nearly tripled in this medium compared to a nitrate-poor media. This made it an interesting candidate for testing its role in the synthesis of bimetallic silver and gold nanoparticles.

Design

We obtained the napA gene from the ASKA collection, where the gene is in the plasmid pCA24N. This plasmid was then transformed into E. coli BL21. The strains were induced with IPTG to express NapA.

Characterization

We set up an experiment to test NapA’s ability to form bimetallic nanoparticles in a cell-free system. In this set up we grew the strains in Mueller Hinton broth (MH broth) and either lysed the cells or spun them down and used the supernatant for the production of nanoparticles. The napA strains were compared to the control of E. coli BL21 strains (WT). The silver and gold was added under the form of AgNO₃ and HAuCl₄ salts. The absorbance was measured after 24 hours.

Fig. 1. Absorbance graph obtained at 800 nm after addition of gold and silver ions to Mueller Hinton (MH) broth media, BL21 supernatant, MH broth with NapA lysate and a combination of NapA lysate and BL21 supernatant. These readings were taken at 24h after the addition of gold and silver salts.

The graph shows absorbance obtained for NapA at 800 nm. As can be observed in the graph, the highest recorded absorbance is for the samples containing medium with salts. This can be attributed to the presence of tryptone in the medium which acts as a strong reducing agent. However, upon the addition of lysate to supernatant with gold and silver salts, the absorbance value drops. A low absorbance is also recorded for lysate with silver and gold.

In a second experiment it was tested what effect the addition of nitrate in the medium has on nanoparticle formation. Here, the grown bacteria were spun down and then the supernatant was used, AgNO₃ and HAuCl₄ salts were added and the reaction was run over 24 hours. The napA strain was compared to only the medium and the WT.

Fig. 2. Absorbance graph obtained at 800 nm for NapA samples in MH broth media and MH broth nitrate. The absorbance was measured 24 hours after the start of the experiment.

The graph shows the formation of nanoparticles in MH broth with and without the addition of nitrate. In only the medium it can be seen that the strains where napA is overexpressed the absorbance is higher, indicating a higher yield in nanoparticles, while it is the opposite in the nitrate rich media. Supposedly, NapA should have a high affinity for the nitrate and thus perform better in a nitrate rich medium. However, take note that the error bars are quite large for MH broth with nitrate, indicating a high variance in between samples. Nonetheless, this shows that NapA is capable of forming nanoparticles, but that maybe lysing the cells is not the most optimal method for a cell-free system. You can find more information on this on the wiki page from the Binanox team.

References

1. Khodashenas, B. Nitrate reductase enzyme in Escherichia coli and its relationship with the synthesis of silver nano particles. Journal of Research in Science, Engineering and Technology 3, 26–32 (2019).

2. Sparacino-Watkins, C., Stolz, J. F. & Basu, P. Nitrate and periplasmic nitrate reductases. Chemical Society Reviews vol. 43 676–706 Preprint at https://doi.org/10.1039/c3cs60249d (2014).

3. Chen, A., Keitz, B. K., & Contreras, L. M. (2018). Biological links between nanoparticle biosynthesis and stress responses in bacteria. Mexican journal of biotechnology, 3(4), 44-69.

Sequence and Features