Team:Dalhousie Halifax NS/Results

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Results

VanA Biobrick Cloning

Colony growth evidence

Our team was able to successively transform and verify the VanA promoter gene of the vanilate operon into DH5α. This was first confirmed by the presence of white glossy colonies, typical of DH5α cells, on a chloramphenicol 25 LB agar plates. The presence of these cells is one piece of evidence that shows a positive indication of successful cloning due to the chloramphenicol antibiotic resistance gene, present in the pSB1C3 genome, allowing cell survival. Figure 1 and Figure 2 display the colony evidence collected. Figure 1 shows our control plate where no colonies have grown, this is what is expected as the chloramphenicol antibiotic resistance gene should not be present in these plasmids. Figure 2 shows the transformed E. coli colonies successfully grown on the chloramphenicol 25 LB agar.

Fig. 1:Control chloramphenicol 25 LB agar plate showing result of DNA transformation pSB1C3 containing no DNA insert after overnight incubation at 37°C. No colonies were found on control plate.

Fig. 2:VanA promoter gene transformation of insert cloning into pSB1C3 vector on chloramphenicol 25 LB agar plates after overnight incubation at 37°C. White E. coli colonies were found.

Gel electrophoresis evidence

By running the mini prepped and digested product of the selected colony DNA and finding two bands present this provided further evidence to support the successful cloning of the VanA promoter gene into DH5α. Looking at Figure 3 it showed that in lanes 11-14 containing the double VanA digests there were 2 bands found at the expected molecular weight marks. The thicker bands present at a higher molecular weight showed the cut pSB1C3 vector at an expected weight for the 2070 bp sequence. The lighter less visible band circled in Figure 3 displayed the presence of the VanA promoter insert. These were also found at an expected molecular weight for the 137 bp insert. Our controls supported that no contamination was found within the samples and the single and double digest controls confirmed that our enzymes were working properly.

Figure 3: Agarose gel at 0.8% concentration with various digestion combinations displayed and used to confirm enzyme function and presence of VanA promoter insert. Lanes 11-14 showed VanA promoter double digests, two bands were found in all lanes giving supporting evidence that the VanA promoter insert was successfully cloned and transformed into DH5α.

Sequence data evidence

A final piece of evidence to confirm the presence of the VanA gene in our plasmid was the sequencing data. Our sequence data, completed by Genewiz, showed that the expected sequence for the VanA gene was present in the entire genome submitted. Figure 4 shows the promoter sequence insert that was successfully cloned into the pSB1C3 genome. The EcoRI and PstI restriction enzyme sites were shown to be present on either side of the promoter insert. This was submitted as our biobrick.

Fig. 4: VanA promoter gene sequenced by Genewiz, viewed using SnapGene. Confirmed presence of VanA insert pSB1C3.

Pyoverdine Fluorescence

from Pseudomonas fluorescens

In order to replicate the results from Del Olmo et al. 2003, we performed two experiments. The first was to examine the fluorescence of pyoverdine when it interacts with aluminum ions in solution. To achieve this, a 100µL dilution series of Al(SO4)3 was prepared and 100 µL of pyoverdine supernatant extract was added to each well. A plate reader was used to measure the fluorescence (excitation 398nm and emission at 460nm).

The pyoverdine extract came from growing Pseudomonas florescens at 30°C, shaking at 100rpm for two days in M9 media. After growing the cells were spun down and the supernatant was passed through a 0.45 micron filter. Plate reader took initial readings. The measurements were too high and needed dilution to be taken. The solutions were diluted to one-twenty seventh of their original concentration. The diluted measurements show a correlation between the concentration of aluminum in the sample and fluorescence measured at 460nm. This data shows decreasing fluorescence with aluminum concentrations. These results support the findings of Del Olmo’s paper, that pyoverdine bind to aluminum ions in solution.

Fig. 5 Aluminum solutions mixed with pyoverdine measured at 398nm and 460nm

The second experiment we performed examined the effects of iron on the binding of pyoverdine and aluminum. Pyoverdin has been shown to preferentially bind with ionic iron over ionic aluminum (Del Olmo et. Al, 2003). To measure this, a dilution series of FeO₄S*7H₂O was created. Each well contained an amount of aluminum, such that after adding 100 µL of pyoverdine supernatant, each well had a concentration of 1µL Al. The results of the Olmo paper showed that optimal inhibition of pyoverdin occurred at a molar ratio of 4:1 Fe to Al. Our results are shown in Figure 6.

Fig. 4: Effect of Iron on pyoverdine-aluminum fluorescence. 100µL solutions of 1µM Al were prepared from Al(SO₄)₃ with varying concentrations of iron prepared from FeO₄S*7H₂O after preparation 100 µL of pyoverdin supernatant was added to solution. Initial readings were too high to be read so a 27 times dilution was applied to each mixture.

Like our first aluminum experiment, the readings were too high to be measured by plate reader. For measurements to be gather dilution to one-twenty seventh of the original concentration was used. The data gathered shows a negative correlation between the presence of iron and fluorescence observed. Above the ratio of 4, there is a notable decrease in fluorescence, supporting the claim that pyoverdine preferentially binds with ionic iron in solution, causing a decrease in optical activity.

Achievements

Successes

• Proved the successful transformation of the Vanilate promoter gene (VanA) into the pSB1C3 plasmid.
• Showed how the pyoverdine production of Pseudomonas fluorescens is able to fluoresce in the presence of Al3+ ions in water.
• Showed how the presence of Fe3+ ions in water diminish the binding of Al3+ ions in water displayed by the decreasing fluorescence in the presence of both metals.
• Designed gene blocks for 2 pyoverdine genes, PvdH and PvdA
• Designed a promoter complex to clone into shipping vector and transform into E. coli
• Designed repressor complex to clone with Vanilate promoter to complete our operon assembly for cloning into shipping vector and transform into E. coli for repressible Vacillate operon system

Failures

• We were unable to successfully clone the Vanilate repressor (VanR) into our shipping vector
• Our team was not successful in confirming cloning and transformation of the repressor or promoter complex
• Cloning and transformation of pyoverdine gene, PvdH and PvdA, was unsuccessful

Future Directions

We hope to successfully clone the different parts of the vanilate operon into one vector in the assembly order of: VanA (promoter), RBS, PvdH and PvdA (pyoverdine expression genes), terminator, promoter, RBS, VanR (repressor), terminator. This system would work as a repressible operon in Pseudomonas fluorescens to express synthesis pyoverdine in a controlled way. In the presence of vanilate the repressor shuts off and when no vanilate is present it is able to express the pyoverdine genes resulting in its synthesis. By using this system we would be able to collect pyoverdine to use in our test kits. The pyoverdine would be dried and collected on our test kit paper filters containing Fe3+ chelators, a positive control and a negative control for on the spot aluminum testing.

Test Kit

There were many different factors we took into consideration when designing the model for our test kit. The two major factors we focused on were product cost and function. Our model consists of a small plastic tube approximately 10 cm high which contains a removable bottom disc approximately 2 cm high. This disc functions to collect and hold water during testing. The disposable filter paper contains 4 different zones. The first zone the collected water passes through would be an iron chelator, to remove any iron present in the water to allow for maximum aluminum-pyoverdine binding giving a more accurate result as to the aluminum concentration present. The second zone would be the dried and bound pyoverdine. This is where the Al3+ ions bind the pyoverdine present on the filter paper. The binding would release visible fluorescence indicating a certain aluminum level based on the fluorescence in comparison to our positive control. The positive control would represent the highest aluminum concentration possible to measure using the filter paper and the control concentration would be given to users in kit. If pyoverdine zone fluoresces to the equivalent of the positive zone, the concentration would be considered higher than biological threshold value and can indicate action should be taken. The final zone would be the negative control to show zero fluorescence.

For this design we consulted our very own team member Jennifer Allott, who has had experience doing on sight water testing in rivers. Suggestions lead to the development of the collection plate to provide a contained system where the water could sequentially pass through each desired zone. The size was also considered to facilitate travel and on sight location use.

This model would act as an inexpensive preliminary test for aluminum concentrations in water to determine if more expensive measures to alleviate the aluminum levels are necessary. By providing a plastic reusable tube the only disposable portion would be the filter papers, which could be re-purchased separately. Overall this would achieve our goal of providing an improved system for detecting aluminum toxicity in Nova Scotia water sources.