Part:BBa_K4083006
rhlB with SacI and SalI sites
The rhlB gene is responsible for the production of rhamnosyltransferase called RhlB 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. [3] 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 rhamnosyltransferase B (RhlB) catalyzes the reaction between 3-(3-hydroxyalkanoyloxy)alkanoic acid and dTDP-L-rhamnose which forms mono-rhamnolipid [1].
Figure 1. RhlA and RhlB metabolic pathway
Part functionality
We used our assembled rhlB primers to extract the nadE gene. (https://parts.igem.org/Part:BBa_K4083016, https://parts.igem.org/Part:BBa_K4083018). 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 rhlB genes were properly extracted as their bands are located below 1.5kbp which is near the actual size of the nadE gene (1334bp). 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:
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 rhlB.
Analysis of biosurfactnt
Since P. aeruginosa already has rhlB genes, we planned to check to what extent the rhamnolipid production will be increased.
| 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 |
Figure 4. Bioremediation treatment of crude oil contaminated soils with crude biosurfactants from the various production set-ups (electrofermentation) using the different P. aeruginosa strains
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)
After the cold acetone precipitation, the precipitates from each sample were hot air-dried. The resulting dried substances were weighted. The average of the obtained values for each type of strain was calculated and recorded in Table 4.1. The largest mass value was observed in the sample from the P. aeruginosa pRGPDuo2 + nadE, while the lowest mass of the precipitate was yielded from the supernatant of wild-type P. aeruginosa. From the data, it can be observed that the greater production of cell products involving rhamnolipids is correlated with the overexpression of nadE genes. Wildtype P. aeruginosa and P. aeruginosa pRGPDuo2 provided values close to each other. P. aeruginosa pRGPDuo2 does not carry inserted genes; therefore, the production is lower. However, the increased value relative to wildtype can be caused by the additional gene load provided by the plasmid. Strains carrying rhlA and rhlB also show increased production of substances; however, the fact that the yield is lower than from P. aeruginosa pRGPDuo2 + nadE might imply that the overexpression of rhlA and rhlB genes is not enough for the enhancing rhamnolipid production under electrefermentative conditions.
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 XhoI site found at 1172
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 65
Illegal NgoMIV site found at 786
Illegal NgoMIV site found at 899 - 1000COMPATIBLE WITH RFC[1000]
| None |