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Applications of BBa_M36162

The ROSES gene (as our research group has dubbed the RhNUDX1 gene) is involved in the production of geraniol, the main component of the scent profile of fragrant roses. The RhNUDX1 protein dephosphorylates geranyl pyrophosphate (GPP), a precursor of geraniol, to produce geranyl monophosphate. This product can then be converted directly to geraniol with the addition of phosphatase.

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RhNUDX1 Plasmid Preparation

In order to enable E. coli bacteria to produce the RhNUDX1 actuator protein, we placed the RhNUDX1 gene described here behind a rhamnose inducible promoter in a high copy plasmid with kanamycin resistance. We also attached a His tag to the end of the gene to enable Western blot detection of the RhNUDX1 part.

RhNUDX1 E. coli Preparation

We followed conventional transformation protocols to introduce a plasmid containing the RhNUDX1 gene into chemically competent NEB5α E. coli and selected for our transformed bacteria with LB media containing kanamycin. E. coli transformed with the RhNUDX1 plasmid exhibited good growth, indicating that there did not seem to be any fitness cost.

Western Blot Confirmation of RhNUDX1 Expression

In order to confirm that our E. coli cells were expressing the RhNUDX1 enzyme, we conducted standard SDS-PAGE Western blotting using a His tag antibody to specifically bind the His epitope appended to the end of the RhNUDX1 sequence. We induced expression of the RhNUDX1 protein with 0.5 mM and 1 mM rhamnose to activate the rhamnose-inducible promoter driving expression of RhNUDX1 on our plasmid. We also included negative controls of untransformed NEB5α E. coli and transformed RhNUDX1 NEB5α E. coli induced with no rhamnose to show that expression of RhNUDX1 is successfully driven by the presence of rhamnose.

In Figure 1, we see that our negative controls exhibited no expression of the RhNUDX1 protein, as bound by the attached His tag. We see expression of RhNUDX1 protein around the expected molecular weight of 16.8 kDa (150 amino acids) in RhNUDX1 E. coli induced with 0.5 mM rhamnose, and we see increased expression of RhNUDX1 protein in the sample induced with 1 mM rhamnose. Thus, we concluded that we had successfully engineered E. coli to express the RhNUDX1 protein when induced with rhamnose.

GPP Toxicity Assay

We proceeded to conduct experiments looking at the function of the RhNUDX1 protein. As GPP is the necessary substrate catalyzed by RhNUDX1 to proceed down the geraniol-producing pathway, we conducted a toxicity assay to determine what concentrations of RhNUDX1 would be lethal to E. coli growth. We selected logarithmic concentrations of GPP between 0-100 μM and incubated RhNUDX1 E. coli in M9/kanamycin media for 24 hours before measuring the OD600.

As shown in Figure 2, we saw that at 100 uM GPP, the OD600 (indicating approximate cell density/concentration) of RhNUDX1 E. coli was approximately the same as the OD600 of the blank M9 sample. This indicated that GPP concentrations in the approximate region of 100 μM are toxic to cells. This informed our future choices with regards to the concentrations of GPP capable to be incubated with our cells for a scent assay.

GPP Scent Assay

Once we had determined suitable incubation concentrations of GPP with RhNUDX1 E. coli, we proceeded to attempt to use the described RhNUDX1 pathway to catalyze the synthesis of geraniol (the basis of rose scent) from GPP. Bacteria for this experiment was cultured in M9/kanamycin media once again in order to avoid the overpowering smell of other bacterial growth media, such as LB. Expression of the RhNUDX1 gene was induced with 1 mM rhamnose, and RhNUDX1 E. coli were incubated with GPP at concentrations of 0 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM. After 48 hours of growth in M9, bacteria were pelleted and the supernatant was harvested, with the expectation that geranyl monophosphate would have been released into the supernatant. The supernatants were then treated with calf intestinal alkaline phosphatase for 1 hour in order to enzymatically remove the remaining phosphate on geranyl monophosphate and convert it into the molecule geraniol. We then wafted the scents of each sample and compared it to the scent of purchased pure geraniol.

Although this experiment was quite qualitative, we determine it could potentially be used as primitive proof of the activity of RhNUDX1. When we wafted the scents of the experimental supernatant samples, it seemed that there was increasing strength of a scent similar to geraniol as GPP concentration was increased. In addition, we repeated the same setup with normal chemically competent E. coli cells. In those samples, we detected the scent of some other compound that smelled differently but was distinctly present. However, we hypothesize that this was an artifact of alkaline phosphatase affecting some other compound naturally secreted by E. coli.

Through the experiments shown here, we have demonstrated the possibility of introducing the RhNUDX1 actuator part into E. coli to allow them to produce the RhNUDX1 enzyme. We have also preliminarily shown that E. coli expressing RhNUDX1 can catalyze the conversion of GPP into geranyl monophosphate or some downstream compound on the way to the production of geraniol. The combination of RhNUDX1 catalysis and alkaline phosphatase appear to be sufficient to synthesize geraniol from geranyl pyrophosphate in bacteria, an unexplored possible method of creating rose scent.

Stanford Location

Plasmid Name: RhNUDX1 DNA 2.0 Gene #: 231832

Organism: E. coli

Device type: Actuator

Barcode #s of glycerol stocks:

0133011806

0133027101

0133027014

Box Label: BioE44 F15 Box 2