Part:BBa_K4083004

nadE gene of Pseudomonas aeruginosa

nadE from Pseudomonas aeruginosa is coding for NAD synthetase which synthesizes NAD+

Usage and Biology

Our team extracted nadE gene from P. aeruginosa to add it into novel plasmid called pRGPDuo2 for P. putida. Apart from nadE gene, planned to add rhlA and rhlB genes that are responsible for rhamnolipid synthesis. Thus, we predicted that dual expression of NAD synthetase and Rhamnosyltrnasferse can allow P. putida to express rhamnolipids in higher rates.

Pseudaminas aeuriginosa - is gram negative bacilus and opportunistic pathogen. NH(3) dependent NAD-synthetase converts deamido-NAD+ to NAD+ by ATP-dependent amidation [1]. ATP + deamido-NAD+ + NH4+ -> AMP + diphosphate + H+ + NAD+

The NAD+ is important for metabolism in organisms. NAD+ can reduce into NADH during cell digestion like glucolysis or Krebs cycle. Thus, more available NAD+ can lead to faster substrate catabolism. Moreover, NADH interacts with the electron transport chain where it releases one electron and one proton. As an electron moves, more protons exit the bacterial membrane which increases the proton gradient. Finally, to reach equilibrium, protons enter the cell by ATP synthase, and one proton can generate up to 3 ATP molecules this way. Thus, one NADH that releases one proton can generate 3 ATP molecules. Finally, NAD+ can interact with NAD kinase to convert into NADP+ which plays a direct role in biosynthesis of rhamnolipids. [2]

3D model from PDB of NH(3)-dependent NAD(+) synthetase from Pseudomonas aeruginosa [3]. The picture was made in Jmol 14.31.57.

Part functionality

We used our assembled nadE primers to extract the nadE gene. (https://parts.igem.org/Part:BBa_K4083019, https://parts.igem.org/Part:BBa_K4083020). Obtained genes were amplified in a PCR machine. Then, these PCR products were analyzed in gel electrophoresis:

We additionally conducted another gel electrophoresis:

Figure 1.1 Gel electrophoresis of PCR products.

Key:

E - nadE for restriction digestion with nhe1 (insert for MCS2)

It can be observed that nadE genes were properly extracted as their bands are located below 1kbp which is near the actual size of the nadE gene (883bp). The smears in each well can result from the high concentration of primers, we learned from our mistake and tried to lower the concentration.

Next, these gels were eluted, and collected genes were inserted into the pRGPDuo2 plasmid. To incorporate nadE genes, we digested plasmids with NheI, SacI, SalI restrictases, and T4 ligase. These plasmids with incorporated nadE gene were electroporated into Pseudomonas putida and Pseudomonas aeruginosa. Unfortunately, due to the lack of time from the COVID-19 situation and late reagents delivery, we were not able to properly insert our genes into P. putida. However, we managed to cultivate P. aeruginosa in kanamycin in LB agar. Then, we extracted these engineered plasmids, and double digested them by SacI and SalI restrictases:

In this picture, C well contains pRGPDuo2+nadE which was double digested. The base pair length corresponds to the actual length of pRGPDuo2 and nadE.

To analyze the effect of NAD on rhamnolipid production, we tested the electro fermentation experiment and analysis of biosurfactants.

Genetically modified P. aeruginosa was introduced to the minimal salt media with crude oil and incubated it for 24 hours. Bioelectrochemical experiments were conducted using the chronoamperometric (CA) method and cyclic voltammetry (CV).

Cyclic voltammetry (CV) method

Reference

[1] UniProt. (n.d.). nadE - NH(3)-dependent NAD(+) synthetase - Pseudomonas aeruginosa (strain PA7) - nadE gene & protein. https://www.uniprot.org/uniprot/A6VD32

[2] Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. New York: Garland Science.

[3] UniProt. (n.d.). nadE - NH(3)-dependent NAD(+) synthetase - Pseudomonas aeruginosa (strain PA7) - nadE gene & protein. https://www.uniprot.org/uniprot/A6VD32 Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 208
  Illegal XhoI site found at 475
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 400
  Illegal NgoMIV site found at 470
  Illegal NgoMIV site found at 691
  
• 1000
  COMPATIBLE WITH RFC[1000]