Part:BBa_K4083007
rhlA gene with SalI and SacI sites at ends
rhlA gene codes for RhlA which is 3-(3-hydroxydecanoyloxy) decanoate synthase in Pseudomonas aeruginosa.
Usage and Biology
P. aeruginosa is a gram-negative bacillus and opportunistic pathogen. It secretes rhamnolipids - the rhamnose containing glycolipid biosurfactant. These biosurfactants are used by P. aeruginosa to emulsify the oil substances for easy digestion. Thus, rhamnolipids can increase the availability of fats which can be important in many different areas like petroleum, bioremediation, cosmetics, food, agriculture, etc. [1] However, due to the toxicity and infectiousness of P. aeruginosa, other alternative organisms are tested. Currently, genetically engineered Pseudomonas putida has more promising results than others. P. putida only lacks two enzymes for mono-rhamnolipid production: RhlA and RhlB. These enzymes are encoded by rhlA and rhlB coding regions in rhlAB operon. It was previously thought that rhlA and rhlB forms heterodimer, however, further research showed that they act independently from each other [2].
Our team planned to extract rhLA and rhlB genes from P.aeruginosa and to insert them into pRGPDuo2 plasmid obtained from Gauttam, R. [2] We developed the new approach to increase the P. putida's rhamnolipid synthesis by adding nadE gene which encodes NAD synthetase. This way, we hoped to see more rhamnolipid production in engineered P. putida.
The RhlA catalyzes the production of 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) from β-hydroxydecanoyl-ACP. HAA is the precursor for rhamnolipid production which is catalyzed by RhlB to form mono-rhamnolipids [1].
Figure 1. RhlA and RhlB metabolic pathway
Part functionality
Visualisation
We used our assembled rhlA primers to extract the rhlA gene. (https://parts.igem.org/Part:BBa_K4083014, https://parts.igem.org/Part:BBa_K4083015). Obtained genes were amplified in a PCR machine. Then, these PCR products were analyzed in the gel electrophoresis experiment:
Figure 2. Gel electrophoresis of PCR products.
It can be observed that rhlA genes were properly extracted as their bands are located near the 1kbp which is near the actual size of the nadE gene (932bp). 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 rhlA 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:
Figure 3. Gel Electrophoresis of extracted plasmids with genes
In this picture, E well contains pRGPDuo2+rhlB which was double digested. The base-pair length corresponds to the actual length of pRGPDuo2 and rhlA.
Analysis of biosurfactnt
Since P. aeruginosa already has rhlA genes, we planned to check to what extent the rhamnolipid production will be increased.
Figure 4. Analysis of emulsification assay results after 24 hours of incubation
A: Treatment with P. aeruginosa Wild type crude biosurfactant (from conventional fermentation) B: Untreated control C: Treatment with P. aeruginosa nadE crude biosurfactant (electrofermentation) D: Treatment with P. aeruginosa rhlA crude biosurfactant (electrofermentation) E: Treatment with P. aeruginosa rhlB crude biosurfactant (electrofermentation)
Table 1. Bioremediation treatment of crude oil contaminated soils with crude biosurfactants from the various production set-ups (electrofermentation) using the different P. aeruginosa strains
| P. aeruginosa Wild Type | Untreated control | P. aeruginosa with pRGPDuo2 + nadE | P. aeruginosa with pRGPDuo2 + rhlA | P. aeruginosa with pRGPDuo2 + rhlB | |
| Emulsification index, % | 39.53 | - | 74.23 | 33.52 | 36.71 |
The emulsification index was identified using the ImageJ software. The remaining crude oil on top of the media of untreated control was taken as a control. Compared to the given value, the emulsification index was identified for other strains of P. aeruginosa. The emulsification index for wild-type P. aeruginosa (39.53%), P. aeruginosa rhlA (33.52%), and P. aeruginosa rhlB (36.71%) was approximately similar, indicating the fact that P. aeruginosa already contains both nadE and rhl genes and electrochemically active. Furthermore, it indicates that overexpression of any gene would not lead to the consequent increase of rhamnolipid production.
However, as the results have shown, overexpression of the nadE gene increased the emulsification index 1.88 times, from 39.53% to 74.23%. It is possible to conclude that the nadE gene overexpression in P. aeruginosa was able to increase the electron pull of the bacteria and elevate rhamnolipid production.
Reference
[1] Chong, H., & Li, Q. (2017, August 5). Microbial production of rhamnolipids: opportunities, challenges and strategies. Microbial Cell Factories. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0753-2
[2] Wittgens, A., Kovacic, F., Müller, M. M., Gerlitzki, M., Santiago-Schübel, B., Hofmann, D., Tiso, T., Blank, L. M., Henkel, M., Hausmann, R., Syldatk, C., Wilhelm, S., & Rosenau, F. (2016). Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Applied Microbiology and Biotechnology, 101(7), 2865–2878. https://doi.org/10.1007/s00253-016-8041-3
[3] 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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 94
Illegal BamHI site found at 654
Illegal XhoI site found at 830 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |