Inulin Staining

Introduction:
The goal of this experiment is to test the inulin production of our bacteria containing one of two plasmids (pJET with either SST or FFT). We are conducting this experiment because we observed weird sugar looking precipitations in our growth medium, and wanted to test whether our FFT were able to produce inulin without SST. As we observed on the growth curves, FFT grew just as fast as E. coli top 10 which indicates that FFT are not able to produce inulin without the presence of SST. This makes sense because SST are producing the platform of 1-kestose that FFT uses to polymerize.
Our hypothesis for this experiment is, based on the growth curves and theoretical knowledge of the enzymes, that FFT should not produce inulin. Though FFT might be able to produce small amounts of the inulin if the sample contains small amounts of 1-kestose.

Method:
This method is purely developed by the students and has been based on chemical properties of Inulin and articles describing inulin isolation from plants.
The idea of this method is to crystallize inulin by adding ethanol. This should happen around 70 % ethanol. The crystals can then be colored by adding phloxine (red color). By washing away the rest of the sample with ethanol the crystals should stay in the filter, which later can be dissolved in water and give a liquid sample that can be measured in the spectrophotometer.

Protocol:
1) Extract 1 mL of bacterial overnight culture (ONC) (make sure to take negative, positive and duplicate samples) to the positive sample add 500 uL of 1mg/mL inulin-solution (add 500 uL H2O to the rest of the samples to ensure equal concentration)
2) Centrifuge samples at 14100 RPM for 2 min
3) Take 1 mL of supernatant and transfer to a falcon tube containing 10 mL ethanol.
4) Add 110 uL of 100 mg/mL phloxine solution
5) Vortex sample to ensure equal distribution of color etc.
6) Press the liquid through a sterile filter with gaps of 0.2 um
7) Wash with 10 mL of ethanol
8) Elute the inulin with H2O into a cuvette
9) Measure absorbance in spectrophotometer
Note: Be carefull when pressing liquid through the filter, they will easely break.

figure 1

Results
On the picture below you can see the filters of the sterile filters (these were extracted by breaking the plastic compartment in which they were located, which resulted in some of the filters being a bit damaged) We conducted the experiment twice. The negative is less stained than the positive throughout the experiments. Also the FFT is mostly more powerful in color than SST.

It was not possible to get a absorbance measurement of our samples. Read about this and more important notes in the discussion section below

Discussion:
We experienced problems with pressing the liquid through the sterile filters and many of them cracked, which resulted in poor washing of the membranes with ethanol, making it impossible to elute the tests in water. This is compromising the validity of our results, hence making our conclusion weak. Though it’s arguable whether we can trust the results from this project, we can still see a color difference between the positive and negative sample. Also the FFT sample is slightly more colored than the SST sample. This could indicate that FFT have been able to produce a small amount of inulin even though it didn’t have SST to produce the trisaccharide platform. this small amount of inulin production would also explain why bacteria having FFT grew just as fast as the top10 strain.

Conclusion
Since the experiment was flawed, so will the conclusion be. If the results is correct we can conclude that FFT is producing a small amount of inulin, most likely because a small amount of trisaccharides exists in the growth medium (LB-medium).