Results

Resistance

All clusters but FuaABC were successfully isolated by PCR but cloning proved to be more difficult. Initial attempts to ligate the cluster behind BBa_J04500 in psb1C3 and transform it into E. Coli yielded no results. Although colony PCR showed a positive colony in some cases of the FDT cluster, plasmids isolated from these colonies were not of the right size to contain the inserts. Sequencing one of these plasmids with the VR and VF2 primers showed insertion of a small fragment containing the end and start of the primer used to PCR the cluster and a small palindromic sequence of 8bp. (ATCGATGCTA)
Membrane proteins can potentially be toxic when expressed at a too high rate. Therefore the construct was inserted in to a low copy number backbone (pSB3K3 / pSB4K5). Additionally the three genes of the smallest cluster (FDT-123) were separately expressed to see if one of them was the cause of the possible toxicity.
Successful transformants were obtained in pSB4K5 for the whole FDT cluster, FDT-2 and FDT-3 under a pLac promoter. Once again the whole FDT cluster showed up successful on colony PCR, but failed to show the expected results when a restriction digest was done on isolated plasmids. Sequencing showed again inclusion of both the primers and a small palindromic sequence (TAGCATCGAT/ATCGATGCTA).
Given that both FDT-2 and FDT-3 were successfully cloned but the whole cluster or FDT-1 separately did not yield any results, FDT-1 , which has homologues proteins in all 3 clusters is the most likely cause for toxicity.

Fusaric Acid Sensing

To isolate the pp1262 gene and the downstream intergenic region, two promoters containing the RFC10 parts were used in a PCR reaction on the genomic DNA of KT2440 P. putida. The product was ligated into pSB1C3 and transformed into chemically competent E. coli cells. The part was digested again and ligated upstream of several different reporter genes. BBa_J13507, which contains an RBS, mRFP and two terminators was successfully transformed. However, after sequencing it became clear that this BioBrick did not contain mRFP, making it unsuitable for this experiment. BBa_J13504, which contains an RBS, mRFP and two terminators was used for further experiments. This construct was ligated and transformed into E. coli. Sequensing confirmed the successful assembly. This construct should only show green fluorescence in the presence of fusaric acid, and no fluorescence when fusaric acid is absent.
The E. coli cells, containing the construct were grown on agar plates containing different concentrations of fusaric acid. At 25 µg/ml, there was no significant difference between the transformed cells and the control cells. At higher concentrations the cells died, due to their inability to pump the acid out of the cell.
The construct was transformed into a different backbone (pSB1K3) and inserted into electrocompetent P. putida cells. The transformed cells were again grown on plates containing different concentrations of fusaric acid. A slight difference in colour could be observed between the transformed cells and the wild-type cells, indicating promoter is only active when fusaric acid is present.


Figure 2 Both plates contain 4 strains of transformed P. putida, containing the BBa_K1493003. The right plate contains 50µg/ml kanamycin and 50µg/ml, whereas the left plate only contains 50µg/ml kanamycin. A slightly increased fluorescence can be observed in the right plate, indicating the promoter is induced by the fusaric acid.

A growth experiment was performed in a 96-wells plate, containing 0, 10, 25, 50, 100 and 250µg/ml fusaric acid and both transformed P. putida cells and wild-type cells. After 18h, at a concentration of 50 µg/ml fusaric acid, the transformed cells showed more fluorescence than the wild-type cells, while both strains showed no fluorescence when no fusaric acid was added, indicating that the promoter is not active when no fusaric acid is present. After washing with PBS since GFP did not include an excretion tag, the wild-type showed no significant fluorescence, whereas the transformed cells clearly did, indicating that the promoter is functional.


Figure 3 Both P. putida strains are grown in LB medium with 5050µg/ml fusaric acid and washed with PBS. GFP fluorescence was checked for 6 samples of each, at 395nm excitation and 501nm emission. This experiment shows that the promoter is active in the presence of fusaric acid. However, the value of the fluorescence is an arbitrary unit, which means a characterized promoter with a downstream GFP gene should be added to the experiment as a positive control to characterize this fusaric acid induced promoter.

The transformed P. putida cells did not survive a fusaric acid concentration of 100µg/ml or higher, possibly because of overexpression of the inhibitor, which would not only inhibit the activity of the GFP, but also of the original fusaric acid efflux pump in the original genome of P. putida.
A new growth experiment was set up, using M9 medium instead of LB medium. In this experiment both the WT P. putida and J04 P. putida are grown in a 96-wells plate at 0, 15, 30, 45, 60 and 75µg/ml fusaric acid. Furthermore, DH5α E. coli cells (WT and BBa_K741002) were also grown in the same plate, but without fusaric acid. The transformed E. coli has a well characterized promoter with the same GFP gene downstream. By comparing the fluorescence of our fusaric acid induced promoter at different fusaric acid concentrations to this constitutive promoter, a characterization can be performed.


Figure 4 The measurement is based on GFP fluorescence in P. putida at increased concentrations of fusaric acid to prove and characterize the activity of the fusaric acid induced promoter, BBa_K1493000. For comparison, the well characterized pLac promoter (BBa_K741002, uninduced by IPTG, see part registry) was used to quantify the activity of this promoter at different concentrations of fusaric acid. The promoter does not respond to low concentrations up to 170nmol/ml. From 255nmol and up, the activity increases. The maximum measured activity of the promoter is 0.21 RPU at 425nmol/ml.