Team:UM Macau/Demonstrate




The internal and external parts of our team collaborated to design and complete the tests and analysis to generate a practical and comprehensive demonstration for our project. The tests included two parts, realistic water test and bacteria viability test. We met some barriers in the process such as unexpected experiment results, obstacles in water samples acquisition, and limitation of time. We managed to break the barriers and bring about some convincing results. After analysis, we believe that our project can function well and be applied in the real situation.

Figure 1: The figure above shows the performance of our E.coli with the OPHT2 variant of the Surface nanoparticle capturing protein (SNCP) under controlled conditions.

In our laboratory tests, our bacteria and nanoparticle were incubated in a stable environment, phosphate buffer pH7 was used as the solution. From Figure 1, OPHT2 is shown able to capture higher concentration of nanoparticle under 6 minutes incubation time as well as that the capturing amount is significantly different compared to the Wild-type bacteria. For later realistic water sample tests we would gauge our E.coli’s ability to perform similarly to the laboratory conditions via comparing its performance with the wild-type bacteria, which doesn’t have our adhesion system construct; we would look whether the amount of captured nanoparticles by our transformed E.coli is significantly different at the P-value < 0.05, if it is then we deem it that our E.coli works in the real environment sample.

We compared the performance of our E.coli in another environment by collecting water samples from different spots in Macau and made the reaction in that solution rather than our phosphate buffer environment. We collected water samples from three spots in Macau as shown in the pictures below. Three locations were chosen for the collection as labeled as A, B and C in the picture below.

Figure 2. The illustration map for water sample collection spots. A. Inner Harbour, B. Reservoir, C. NamVan-lake.

OPHT2 construct is chosen for our demonstration test because it can emit green fluorescence light which is convenient for us to check eGFP protein expression after we have induced the batch of bacteria with 1mM IPTG.

Figure 3. Tests set-up in the format of the 96-well plate.

Each construct has three technical repeats for each timepoint group as shown in the format above. Supernatant samples were collected and measured the fluorescence amount using a microplate reader.

Figure 4: Performance test of our SANCE in different water samples from different places.

We chose two incubation time points: 6 minutes and 48 minutes to plot the graph.

In sample A obtained from Inner Harbour, we can see the concentration of nanoparticles captured by OPHT2 bacteria and wild-type bacteria has significant differences between each other. This river water sample contains heavy metals molecules and unknown dirty particles which might occupy the nanoparticles binding sites on the sticky protein of our bacteria SANCE. In terms of different time points incubation, OPHT2 shown able to capture more nanoparticles after 48 minutes incubation compared to 6 minutes. From Figure 4A, in fact we can see the concentration of nanoparticle captured by OPHT2 after 6 minutes and 48 minutes do not change, the significant increases due to the decrease of nanoparticle captured by wild-type bacteria after longer incubation time. Therefore, we can conclude that OPHT2 bacteria is able to capture nanoparticles even under the water sample A’s harsh environment due to the fact that there are significant differences in both time points.

In sample B which is a reservoir used to store water, Figure 4B shows that the nanoparticle concentration differences between OPHT2 and wild-type are both significant at 6 and 48 minutes incubation, indicating that our OPHT2 construct works well in sample B. From another viewpoint, the data is more significant under 6 minutes incubation compared to 48 minutes. Along with the increase of incubation time, the nanoparticle binding sites are gradually occupied. Thus, the concentration of nanoparticle captured decreases after 48 minutes incubation. As there are significant differences in both time points, we can conclude that our bacteria with OPHT2 construct works in water sample B environment.

From Figure 4C, it is shown that our bacteria SANCE performed the best capturing ability in sample C which is from the Outer Harbour. The nanoparticle concentration captured by OPHT2 bacteria is way higher than wild-type bacteria at 6 minutes incubation. At 48 minutes incubation time point, the significance decreases compared to the 6 minutes time point as the protein binding sites for nanoparticles will reach a saturation point along with the increase of incubation time. As this water source is from lake water, it makes sense that our bacteria SANCE can perform its sticky function normally in a cleaner water environment which has related normal pH and stable condition for bacteria in comparison to sample A and B. Therefore, it can be concluded that our bacteria able to capture nanoparticles in water sample C.

Based on the results above, we can safely conclude that our SANCE is able to perform similarly in realistic water conditions.

The video shows our SANCE in action. Our SANCE is in the circled red area and the fluorescent signal in blue is our nanoparticle. We can see from this video that the nanoparticle signal is following the movement of our SANCE. Thus, this is huge evidence of our organism being able to collect nanoparticle targets.

During our interview to Hong Kong experts, one of the interviewees from The University of Hong Kong, Professor Leung, Kenneth Mei Yee raised a concern that toxicity of nanoparticles might be harmful and powerful to kill our bacteria organisms. Therefore, we designed a survival test to investigate whether toxic nanoparticles can actually kill our bacteria. We have chosen two nanoparticles which is AgNP (20nm in size) and ZnO (30±10nm) respectively. 5 different concentrations of Zinc Oxide and Silver nanoparticles were prepared in LB broth solution with concentrations of 0.5 mg/L, 1 mg/L, 20 mg/L, 40 mg/L and 60 mg/L respectively. We chose this range because according to literature, the average concentration of AgNP in waste water treatment plants is 1 mg/L.

Figure 6. The test set-up for 96-well plate for survival analysis of SANCE under real water samples with AgNP and ZnO.

In both Figure 7 and 8, we can observe that the Optical Density (OD) of bacteria rises when incubation time increases up to 12 hours as bacteria keeps growing along with time. In terms of nanoparticle concentration, both graphs present a similar trendline. The OD value still maintain great even the concentration of nanoparticles (AgNP and ZnO) increases up to 30mg/L. This indicates that our bacteria SANCE can tolerate the toxicity of nanoparticles which is smaller than our bacteria and able to survive even in high concentration of nanoparticles.

Figure 7. OD value of bacteria after incubation with 6 different concentrations of AgNP nanoparticles under 5 different incubation time length

Figure 8. OD value of bacteria after incubation with 6 different concentrations of ZnO nanoparticles under 5 different incubation time length



iGEM 2019 UM_Macau