Part:BBa_K4083008
RPGDuo2 plasmid with nadE and rhlBA genes
Our team added rhlA/rhlB and nadE genes to pRGPDuo2 plasmid. The pRGPDuo2 plasmid (https://parts.igem.org/Part:BBa_K4083000) was obtained from Gauttam, R. [1] We planned to insert rhlA and rhlB genes to MCS1 and nadE gene to MCS2 of pRGPDuo2 plasmid. Thus, the rhlA and rhlB genes are controlled by the IPTG-inducible Ptac promoter, while the nadE gene is controlled by aTc-inducible PtetR/teA promoter. Those promoters are regulated by lacI and tetR repression systems
Structure of Plasmid
This plasmid consists of four parts: pRGPDuo2 plasmid; nadE, rhlA, and rhlB coding sequences (https://parts.igem.org/Part:BBa_K4083000, https://parts.igem.org/Part:BBa_K4083004, https://parts.igem.org/Part:BBa_K4083006, https://parts.igem.org/Part:BBa_K4083007). However, for Biobricks construction purposes, we segmented pRGPDuo2 plasmid into smaller fragments can be accessed below in Sequence and Features part
The general Biobricks construction would look like this:
https://static.igem.org/mediawiki/parts/e/eb/BBa_K4083008-biobricks.tif
By inserting this plasmid, we predicted the effective rhamnolipid production by Pseudomonas putida. Since Pseudomonas putida has all genes required for rhamnolipid production except for rhlA/B, the rhlA and rhlB coding sequences inserted in plasmid can allow engineered P. putida to synthesize mono-rhamnolipid by itself. [2]
Part functionality
While in the beginning it was presumed that we will construct a plasmid with three genes incorporated into it, we were not able to do so. Instead, we inserted nadE, rhlA, rhlB genes into pRGPDuo2 separately. The reason for it is that in the beginning we designed incorrect primers and were not able to extract the rhlA/B gene from P. aeruginosa. However, as a future prospect, we will work further on optimizing the extraction and ligation of genes of interest.
Figure 1. Gel electrophoresis image of double digested plasmids
Most of the experiments were dedicated to the insertion of nadE, rhlA, rhlB genes separately into the RGPDuo2 plasmid. To prove that we successfully introduced genes of interest into plasmid, we performed double digestion of engineered plasmids with restriction enzymes SacI and SalI. While RGPDuo2 plasmid (A) weighs 7928 bases, in reality, it ran less distance possibly due to its nicked form. Cutting of plasmid with restriction enzymes resulted in its linearized form (B), which acts as a reference band and which normally migrates more than uncut one. The digested engineered plasmid will be visualized with two bands- first for vector DNA and second for the gene of interest. Therefore, here we have bands slightly less than 8kb and those for corresponding genes: nadE with 883 bases (C), rhlA with 932 bases (D), rhlB with 1334 bases (E).
Bands from B to E represent products of engineered plasmid digestion, which was performed with restriction enzymes SacI and SalI using NEBuffer r1.1 for 1 hour at 37°C, followed by heat inactivation at 65°C for 20 min.
Bands from F to I depict the same products with the only difference in time of digestion - 15 minutes for restriction digestion reaction. However, due to insufficient digestion time, we were not able to get the nadE band.
Bands on the gel electrophoresis image correspond to the following samples: A - pRGPDuo2 plasmid uncutted B - pRGPDuo2 cut with SacI and SalI (digestion at 37° for 1hour) C - pRGPDuo2 + nadE digested with SacI and SalI (digestion at 37° for 1hour) D - pRGPDuo2 + rhlA digested with SacI and SalI (digestion at 37° for 1hour) E - pRGPDuo2 + rhlB digested with SacI and SalI (with digestion at 37° for 1hour) F - pRGPDuo2 cut with SacI and SalI (digested at 37° for 15 min) G - pRGPDuo2+ nadE digested with SacI and SalI (digested at 37° for 15 min) H - pRGPDuo2+ rhlA digested with SacI and SalI (digested at 37° for 15 min) I - pRGPDuo2+ rhlB digested with SacI and SalI (digested at 37° for 15 min)
Although we did not have enough time to conduct electro fermentative experiments with genetically engineered P. putida, we inserted our plasmids into P. aeruginosa to induce overexpression of nadE, rhlA, rhlB genes.
Cyclic voltammetry (CV) and chronoamperometric method (CA) showed that P. aeruginosa with overexpressed nadE (red line) was found to be the most electrochemically active. This can be explained by the increased number of 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 higher yield of desired biosurfactants.
Figure 2. 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).
Figure 3. Chronoamperometry data 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)
Reference
[1] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514
[2] Tiso, T., Sabelhaus, P., Behrens, B., Wittgens, A., Rosenau, F., Hayen, H., & Blank, L. M. (2016b). Creating metabolic demand as an engineering strategy in Pseudomonas putida – Rhamnolipid synthesis as an example. Metabolic Engineering Communications, 3, 234–244. https://doi.org/10.1016/j.meteno.2016.08.002
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 5565
Illegal BamHI site found at 6125
Illegal XhoI site found at 1757
Illegal XhoI site found at 2024
Illegal XhoI site found at 6301 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1949
Illegal NgoMIV site found at 2019
Illegal NgoMIV site found at 2240
Illegal NgoMIV site found at 4400 - 1000COMPATIBLE WITH RFC[1000]
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