Introduction
We believe that everyone has the right to ensure food security, so we design a product which contains E. coli with the gene fragments to detect toxins. We embed E.coli into microcapsules and put them into centrifuge tubes. We have biological safety system. Our product will be kept in P1 lab*. People can send oil samples to the lab, which fastens the inspection and provides scientific clues for the government when inspecting illegal industries. The embedding of E. coli saves cost and time and has high-efficiency detection mechanisms.
*125 universities in Taiwan have P1 labs, and 66 universities in northern Taiwan have P1 labs.
Safety-Triple biosafety certificated mechanisms
The first and foremost risk that our final product: microcapsules with genetic-modified E-coli will pose is that E-coli might leak out of the tube and further contaminate the environment. Our work on biosafety issue is to reduce the chance that the E-coli will leak out and contaminate the environment. We have developed triple biosafety certificated mechanisms that will reduce the risk.
- Microcapsule
First mechanism is that we will package the E-coli biosensors into microcapsules that prevent E-coli within from leaking out.
- Chitosan
Second mechanism is that we have selected one chemical compound- percarbonate that can carry out the ability to sterilize and kill 99% E-coli once activated.
- Sodium percarbonate
Third mechanism is that after using this product, one can pour the alimentary acetic acid into the product to activate the sterilizing procedure.
Because the alimentary acetic acid can dissolve both layers of chitosan and activate percarbonate for sterilizing. For those three safety mechanisms, we hope to reduce the potential risk that our product might posed and thus can be further utilized by people all over the world.
Experiment Design
1. Immobilization
1.1 Compare the effect of different immobilization methods on microcapsules’ patterns
We chose three methods to immobilize E.coli. Then we made a pre-test to ensure each method’ feasibility, including immobilization materials and equipment.
1.1.1Procedure
PVA(Polyvinyl alcohol)-SA(Alginate)
- Pour some solution into beakers.
- Draw certain amount of 8%PVA-1%SA and add it into 3%BA(Boric acid)-1%CaCl2.
SA(Alginate)
- Pour some solution into beakers.
- Draw certain amount of 8%PVA-1%SA and add it into 3%BA(Boric acid)-1%CaCl2.
ACA(Alginate & Chitosan)
- Pour some solution into beakers.
- Draw certain amount of 1.5%SA and add it into 100mmol/LCaCl2
- Throw the microcapsule into 0.3%Chitosan.
- Throw the microcapsule into 0.15%SA.
- Use syringe to inject sodium citrate in the microcapsule.
1.1.2 Result
1. Different materials of immobilization
We decided to apply only SA and ACA materials to our experiment, and we will delete step e. of ACA because it’s infeasible.
2. Comparison of syringe and pipet
We decide to apply syringe to our experiment because syringe has a better effect on microcapsules’ patterns than pipet.
1.2 Compare the effect of different preserving temperatures of microcapsules on E.colis' survival
We chose three common preserving temperatures to carry out our experiment, including room temperature 25℃, cooler compartment 4℃, and freezer compartment -18℃.
1.2.1 Procedure
- Embed the bacterial liquid and preserve it in -18/4/25℃
- Break and dissolve cell-loaded microcapsules with microcapsule-broken solution every 48 hours.
- Draw bacterial liquid and culture on the solid medium.
- Put them in 37℃ for 48 hours.
- Count the amount of E.coli
1.2.2 Result
WE HAVEN’T FINISHED
2. Freeze-dried & Heavy metal absorption of chitosan
2.1 Compare the effect of freeze-dried and oven-dried chitosan on heavy metal absorption
There are many kinds of heavy metal, and here we take Cu(II) ion for example.
2.1.1 Procedure
- prepare 1.5 g of chitosan
- add in 0.4 M 50 ml of citric acid
- use the magnetic mixer at the speed of 100 rpm to mix the mixture for 12~24 hr
- use the peristaltic pump to drop the mixture into 0.9 M NaOH mixture
- wait for 72 hr after the reaction
- after been washed with DI water, put the chitosan-citrate beads into the oven and dry at 35℃ for 24 hr
2.1.2 Result
▼Table 1: The effect of freeze-dried and oven-dried chitosan on Cu(II) ion adsorption
Chitosan |
Adsorption(mg/g) |
Adsorption(mg/g) |
Adsorption(mg/g) |
Adsorption(mg/g) |
temperature |
30℃ |
40℃ |
50℃ |
60℃ |
Freeze dried |
123 |
129 |
131 |
141 |
oven dried |
113 |
115 |
120 |
127 |
2.2 Compare the effect of different initial concentrations on chitosan for heavy metal adsorption
2.2.1 Procedure
- prepare 3.929 g of copper sulphate.
- add DI water to 1000 ml
- take out 200 ml of the mixture, and add DI water to 1000 ml
- add 1 g of freeze dryer-dried chitosan-citrate beads into the different concentration Cu(II) ions mixture, and mix at the speed of 100 rpm
100 mg/L
250 mg/L
500 mg/L
750 mg/L
1000 mg/L
- take samples at 0 hr, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, 48hr, 72 hr
2.2.2 Result
The higher the concentration is, the better the chitosan-citrate beads’ adsorption will be; but the percentage of adsorption will be lower
▼Table 2: The effect of different initial concentrations on chitosan-citrate beads for Cu(II) ion adsorption.
Chitosan-citrate |
Initial concentration of copper(ppm) |
Adsorption(mg/g) |
Percentage of adsorption(%) |
100 |
83 |
83.07% |
200 |
123 |
65.455% |
500 |
196 |
39.272% |
750 |
270 |
36% |
1000 |
308 |
30.857% |