Difference between revisions of "Part:BBa K4083004"

Revision as of 21:49, 21 October 2021

__NOTOC__ BBa_K4083004 short 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+ https://static.igem.org/mediawiki/parts/thumb/4/45/BBa_K4083004-NADS_reaction.png/799px-BBa_K4083004-NADS_reaction.png Figure 1. NADS function The NAD+ is important for metabolism in organisms. NAD+ can reduce into NADH during cell digestion like glycolysis or the 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 the biosynthesis of rhamnolipids. [2] ===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 2 gel electrophoresis experiments: https://static.igem.org/mediawiki/parts/0/01/NadE_emphasized.jpg https://static.igem.org/mediawiki/parts/d/d6/NadE_emphasized1.jpg Figure 2. Gel electrophoresis of PCR products. 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: https://static.igem.org/mediawiki/parts/3/3a/NadE%2Bplasmid_emphasized1.jpg Figure 3. Gel Electrophoresis of extracted plasmids with genes 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. Bioelectrochemical testing 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 https://static.igem.org/mediawiki/parts/thumb/f/fd/Cyclicvoltammetry.png/751px-Cyclicvoltammetry.png Figure 4. Cyclic voltammograms (CV) at 10mV/s scan rate between -400mV and 400mV for the different electro fermentative setups for production of rhamnolipids by the different test P. aeruginosa strains (engineered and wild type). Media used: MSM (minimum salt media) + oil + casein Cyclic voltammetry (CV) plot shows distinct oxidation (upwards) and reduction (downward) curves which imply that the set-ups are redox-active (electrochemically active). Figure 1 depicts that P. aeruginosa with overexpressed nadE (red line) had the highest peak among the test strains after 24 h incubation. This can be explained by the increased respiratory activity of the strain caused by increased NAD synthetase production. This in turn increases metabolic reactions that involve NAD as an electron carrier. It can be hypothesized that such a scenario will lead to faster cell growth of bacteria thereby inducing an increased expression of rhlB and rhlA genes and resulting in a higher yield of desired biosurfactants. Although other strains of P. aeruginosa with overexpressed rhlA and rhlB genes and wild-type strain showed some peaks at similar oxidation-reduction spots, they had less respiratory activity because they did not possess overexpressed NAD synthetase. Therefore, we hypothesized that this increased respiratory activity could be significant in enhanced biosurfactant yields as well. Chronoamperometric (CA) method https://static.igem.org/mediawiki/parts/thumb/6/61/Chromoamperometric.png/800px-Chromoamperometric.png Figure 5. Chronoamperometry data (CA) at 400mV poised potential for the different electro fermentative setups for production of rhamnolipids by the different test P. aeruginosa strains (engineered and wild type). Media used: MSM + oil + casein It can be observed that all the engineered strains yielded higher current outputs than the wild type strain with highest current generated by the P. aeruginosa nadE set up. Total electrical charge in Coulombs produced after 24 h incubation by the different test setups carrying the different strains is shown above. The graph depicts that P. aeruginosa nadE setup produced the highest charge of 10.8 mC at 24 h. The trend in total charge production was the following: P. aeruginosa nadE > P. aeruginosa rhlA > P. aeruginosa wild type > P. aeruginosa rhlB. ===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 BBa_K4083004 SequenceAndFeatures