Part:BBa_K653000

Re-designing and re-assembling of the rhamnosyltransferase BioBrick part: "The Biosurfactator"

This year`s BioBrick construction effort will be due to a re-designing and re-assembling of our last year surfactant BioBrick project and improving with a new concept in which we degrade hydrocarbons as well as using the surfactant BioBrick, that we will describe next.

Pseudomonas aeruginosa species of bacteria produce the natural rhamnosyltransferase gene complex (RhlAB). This is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid, which is a biomolecule with surfactant properties, but the natural RhlAB gene has illegal restriction sites that make it incompatible with the Assembly Standard Protocol 10, specifically the Pstl1 restriction sites.

The design and construction of the rhamnosyltransferase gene into a BioBrick part was the main goal and achievement of the iGEM Panama Team 2010, but since the beginning of this year’s iGEM project we failed in producing the rhamnolipid compound into our E. coli based-factory, because our last year’s BioBrick appeared to have been denaturalized. We then ordered our BioBrick part BBa_K424018 (iGEM Panama team 2010) from The Registry and once it arrived we ran some tests and we noticed that the part was no longer fitted into the plasmid backbone.

As scientist and iGEMers, we have the responsibility to deliver a functional BioBrick, thus according to iGEM’s competition new slogan, "Quality not Quantity", we have embraced the challenge of re-designing and re-assembling the same BioBrick part from last year in order to comply this new standard based on quality. So we bring the Biosurfactator!!!

Further Characterization by ColumbiaU_NYC iGEM 2016 team

Rhamnolipids, a class of glycolipids characterized by a rhamnose moiety attached to a fatty acid tail, is produced by many organisms—with the Pseudomonas aeruginosa as the most predominate. We have shown that Pseudomonas putida produces both mono-rhamnolipids and di-rhamnolipids with the addition of the rhlAB and rhlC operons, respectively. Previous research has shown that di-rhamnolipids repel the Aedes aegypti mosquito. We have shown that both di-rhamnolipids and mono-rhamnolipids repel Aedes aegypti mosquitoes. We have also shown that rhamnolipids are compatible with human keratinocytes in the presence of both Pseudomonas aeruginosa and Pseudomonas putida. Lastly, we have shown that rhamnolipids are compatible with Staphylococcus epidermidis—a skin microbiome organism.

Introduction

Rhamnolipids are a class of glycolipids characterized by a rhamnose moiety and a fatty acid tail. While rhamnolipids are produced in a variety of organisms, Pseudomonas aeruginosa is most frequently cited. In Pseudomonas aeruginosa, genes rhlA and rhlB are cooperative to form the complex rhlAB that codes for the enzyme rhamnosyltransferase 1. The enzyme rhamnosyltransferase 1 catalyzes the addition of a (hydroxyalkanoyloxy) alkanoic acid (HAA) fatty acid tail to a rhamnose sugar to produce a mono-rhamnolipid. Similarly, rhlC codes for the enzyme rhamnosyltransferase 2, which catalyzes an addition of another rhamnose moiety to a mono-rhamnolipid to form a di-rhamnolipid.

Rhamnolipids are predominantly known for their biosurfactant properties, which possesses industrial applications ¹. Di-rhamnolipids have also been shown to repel the Aedes aegypti mosquito ². In our investigation, we have confirmed with statistical significance that di-rhamnolipids repel Aedes aegypti. We have also shown with statistical significance that mono-rhamnolipids repel Aedes aegypti. The compatibility of rhamnolipids with human skin was also a main concern of ours—as rhamnolipids have been shown to be a virulence factor. We have shown that rhamnolipids are compatible with human keratinocytes in the presence of both Pseudomonas aeruginosa and Pseudomonas putida. Likewise, we have shown that rhamnolipids are compatible with Staphylococcus epidermidis—a skin microbiome organism. Lastly, we have confirmed the both mono-rhamnolipids and di-rhamnolipids are producible in Pseudomonas putida with the addition of rhlAB and rhlC, respectively.

Mutant rhlAB P. putida produces rhamnolipids

Quantification of rhamnolipids

In order to accurately measure the amount of rhamnolipids produced by our mutant strains, we used supercritical fluid chromatography (SFC-MS). First, a test run was executed with a mixture of mono-rhamnolipids and di-rhamnolipids at the concentration of 5 mg/mL by running the sample through the column packed with 2-PIC. From this test run, we have obtained the retention times of mono-rhamnolipids (rha-C₁₀-C₁₀: pseudomolecular ion of 503.56 m/z) and di-rhamnolipids (rha-rha-C₁₀-C₁₀: pseudomolecular ion of 649.8 m/z) to be approximately 3.974 min and 4.942 min respectively. Then, a calibration curve was constructed with 95% pure mono-rhamnolipids, and the limit of detection was found to be approximately 5 µg/mL. The mass fractions were obtained from electrospray ionization (ESI) negative mode.

From our TLC analysis, it was found that supplementing the LB media with glucose is crucial to the production of rhamnolipid. Therefore, for SFC-MS analysis, all the mutant strains (E. coli_H2_RhlAB, E. coli_L1_RhlAB, and P. putida_L1_RhlAB) were grown in LB supplemented with glucose. From the SFC-MS data, it was found that mutant E. coli strain makes more mono-rhamnolipids than mutant P. putida. Furthermore, the promoter strength was confirmed as expected since the mutant E. coli strain transformed with a high expression level promoter H2 produced almost 6 times more rha-C₁₀-C₁₀.

In order to investigate the optimum growth conditions for rhamnolipid by the mutant P. putida strain, the amount of glucose added and the time of growth were varied. Using the calibration curve above, we were able to measure the accurate amount of rhamnolipids produced in each cell culture. From this data, we have concluded that P. putida produces the most mono-rhamnolipids when grown for 24 hours in the media LB supplemented with 50 g/L of glucose.

We have also tested the mutant strain of S. aureus RN4220, the strain that carries shuttle vector for S. epidermidis. Unfortunately, SFC-MS data didn't show any production of rhamnolipids from S. aureus strain.

Contribution from iGEM21_NU_Kazakhstan

Team: iGEM21_NU_Kazakhstan
Author: Arsen Orazbek
Our team developed a project called RemiDuET. We also ColumbiaU_NYC iGEM 2016 team considered engineering P. putida to make it synthesize the rhamnolipids. We wanted to incorporate the nadE gene with rhlA and rhlB genes in the special pRGPDuo2 plasmid that we obtained from Gauttam, R. Also we wanted to enhance the rate of rhamnolipid production by electrofermentative conditions.

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 rhlAB 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 plasmids, 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 uncut
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.

 

For more information, you can look to this page: https://parts.igem.org/Part:BBa_K4083008

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

Mono-Rhamnolipids repel Aedes Aegypti

In order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. Aedes aegypti, the species of mosquito observed to carry Zika virus, were grown from larval stage, and females were sorted at the pupae or adult stage. Since only females consume blood for reproduction, we were only interested in using them for the assays.

One day before experiment, 50 total mosquitos (with 30 females) were isolated in cages and starved from 23-25 hours. Each cage was then taken to a warm room (~30 oC), and the cage was covered with wet paper towels to preserve humidity. For each trial, our blood feeding system (Figure) was placed on top of the cage each with a cotton gauze soaked with either negative control water, 1 mg/mL mono-rhamnolipid solution, 1 mg/mL di-rhamnolipid solution, or positive control 25% DEET, and the mosquito activity was videotaped for 1 hour. Afterwards, the cage was taken to the cold room to paralyze the assayed mosquitoes, and mosquitoes that had consumed blood were counted. It is important to note that the age of female mosquitoes and the time of feeding played an important role in how mosquitoes behave. Typically, it is optimum to use female mosquitoes of age from 4-6 days for feeding assays as any mosquitoes older than this age range will be too old to reproduce, and thereby not needing to drink blood. Furthermore, their feeding is most active 4 hours before dusk. Some of our trials that didn’t meet these criteria did not result in any feeding, but we did observe significant difference in landing between the control and rhamnolipids. Our landing assay results showed that while DEET was the strongest mosquito repellent with no landings or fed mosquitos, 1 mg/mL mono and di-rhamnolipid still showed statistically significant repulsion as shown in the graph below.

P. putida, S. epidermidis, and rhamnolipids are compatible with human keratinocytes

Determination of rhamnolipid IC50

Keratinocytes, human skin cells, were grown for several days. When the cells were 80% confluent, they were seeded in 24 well plates at a density of 2.5105. The cells were weaned off of antibiotics the following day before they were treated with varying concentrations of rhamnolipids and the reagent MTS. The MTS assay reveals the cell viability of the cells. Using this information, the data was normalized and statistically analyzed to determine the keratinocyte IC50—or the concentration of rhamnolipid that induces 50% cell death. The IC50 was determined to be between 45.19 µg/mL and 65.52 µg/mL. Relating the results to rhamnolipid quantification, the concentration of rhamnolipid the construct produces should not cause significant cell death.

Keratinocyte cell viability bacteria assay

Keratinocytes were co-cultured with different strains of bacteria (Pseudomonas putida, Pseudomonas aeruginosa PAK, Staphylococcus aureus, Staphylococcus epidermidis, and mutant rhlAB P. putida). Half were cultured in plain DMEM with serum, and half were culture in DMEM with 1 mg/mL mixed mono- and di- rhamnolipids. After co-culturing, the keratinocytes were washed with PBS, exposed to gentamicin in an attempt to kill the bacteria, and incubated in MTS cell viability assay for up to 4 hours and viewed in a plate reader. MTS assay is colorimetric cell viability assay and reacts with NADPH-dependent dehydrogenase enzymes, which are only active in live (metabolically active) cells³. For the MTS assay, pure media were used as a negative control (100% cell death), and keratinocyte culture with normal DMEM was used as a positive control (“0%” cell death, or the maximum number of cells that could be alive).

The results indicate that there is no consistent trend regarding the addition of rhamnolipid and cell viability. Rhamnolipids did not significantly increase or decrease cell viability regardless of the bacteria type as shown in the first figure since the error bars overlap. We hypothesized that the concentration of P. putida would not influence cell viability as it is an environmental strain not nearly as potent as other bacterial strains such as Pseudomonas aeruginosa PAK. As depicted in the second figure, all MOIs (ranging from 0 to 20) did not significantly influence the cell viability of the strain as shown by the overlapping error bars in the graph. These results overall indicate that our construct may not cause significant cell death once applied to the skin in an acute setting of a few hours.

Rhamnolipids are compatible with Staphylococcus epidermidis

In order to make sure that our S. aureus strain (RN4220) and our S. epidermidis (RP62A, 1457) strains would not be killed by the production of rhamnolipids, we conducted 3 rhamnolipid survival assays with the 1g/L rhamnolipids necessary for mosquito repelling. Kanamycin added to S. epidermidis cell culture was used as a negative control. Although the addition of higher concentrations of rhamnolipids (250 mg/L and above) depressed the growth of all our Staphylococcal species, it didn’t kill the cells but only slowed down the growth.

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¹ Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.
² Silva, Vinicius L., Roberta B. Lovaglio, Claudio J. Zuben, and Jonas Contiero. "Rhamnolipids: Solution against Aedes Aegypti?" Frontiers. Frontiers in Microbiology, 16 Feb. 2015. Web. 23 Oct. 2016.
Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.
³ "MTS Cell Proliferation Colorimetric Assay Kit." BioVision. Web.

Re-designing "The Biosurfactator" The first Central American BioBrick

Assembly Compatibility:

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

  Illegal BamHI site found at 103
  Illegal BamHI site found at 663
  Illegal XhoI site found at 839
  Illegal XhoI site found at 2125
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1018
  Illegal NgoMIV site found at 1739
  Illegal NgoMIV site found at 1852
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 328
  Illegal BsaI site found at 1368
  Illegal BsaI.rc site found at 512
  

How to use it as a BioBrick? :

We developed this standarized part to be inserted into E. coli for rhamnolipid production through the mutation of the gene that is responsible to synthesize the key enzyme rhamnosyltranferase (RhlAB) to be compatible with Assembly Standard 10. We have achieved this by performing several silent mutations using the QuikLighting Multi Site-Directed Mutagenesis Kit from Stratagene. This rhamnosyltransferase BioBrick (Rh1AB_BB) is ready to be tested on an expression plasmid device following the Assembly Standard Protocol 10.

Application of The Biosurfactor :

ITB_Indonesia 2015 team used this coding sequence to make BBa_K1685011 part for rhamnolipid production device under T7lac promoter. The supernatant of IPTG-induced E. coli BL21(DE3) transformant showed surfactant activity comparable to Tween 20%.