Result and Discussion
I. Plasmid Construction
DNA Preparation of Gibson assembly
Fig1 Gel electrophoresis of vectors and insert
To prepare our vectors and inserts for Gibson assembly, we amplified three vectors which are LP vector and LPAg vector from pETD-p-nl-lux backbone, T7 vector from pETD-T7-ag43-lux backbone. Ag insert was amplified from pETD-T7-ag43-lux backbone. Two backbones were brought from company. From the gel run results above, we can see the bands of vectors are around 6000kb, and band of Ag is around 4000bp, which indicates that we got successful amplified products. After checking the gel result, we did gel purification to get those DNA.
Restriction Enzyme Cut
Fig2 Gel electrophoresis of digested plasmids
After performing Gibson assembly of the DspB insert into the LP vector that we amplified previously, we picked a colony and then extracted their plasmid. Then we used a restriction enzyme cut to check resulting band combination. From the gel run results above, we can see two fragments from the plasmid are at around the expected size of 5672 bp and 1715bp. This result shows that we have successfully constructed pETD-P-DspB.
II. Protein Expression Test
Coomassie blue gel stain
Fig3 Coomassie blue gel staining with two different induction time points
Fig4 Protein expression test using Coomassie Blue Staining with 4 hours IPTG induction time
The Coomassie blue staining procedure was utilized to check the expression of our LuxR protein, which would primarily use for detecting AHL molecules. We can see from Fig.3 above that in the 4 hour induced groups, the concentration of IPTG which is 0.1 mM showed bands at the expected protein mass of approximately 28-30 kDA which is a range wherein LuxR which weighs 28.56 kDA is expected to be present.
In order to optimize the band and get a clearer band. We repeated this experiment to double confirm the results. With the following optimizations:
1.Prolonging centrifuge time from 30s to 5mins to collect more bacteria.
2.Measure protein concentration through Bradford assay and dilute the samples to the same concentration.
As expected in Fig4 above, we could see that the LuxR protein is able to be successfully expressed in our BREAC.
Fig5 E. coli BL21 biofilm formation incubation conditions optimization
In order to do proteinase K degradation testing, we first need to find the optimal culturing conditions for the biofilm. It is important to use rounded bottom wells so that the biofilm can be precipitated. The graph shows that under the condition of 72hrs incubation we can get the maximum amount of biofilm.
Fig6 Proteinase K degradation efficiency with different doses
We were supposed to test the degradation efficiency using our enzyme expressed from Engineering E. coli BL21. But due to time constraints and reagent availability during the pandemic, we were only able to test it by directly using a proteinase K solution to represent our biofilm degradation capability. Here we can see the biofilm can be degraded effectively under condition of 37°C, 30mins incubation. If had more time, we would optimize the gradient of proteinase K. use 10x of proteinase K stock concentration and make a dilution range from 10^-7 to 10^0, with negative control.
Dspb, DNaseTA and Alkaline serine proteinase are the three target enzymes, we will test all of them and pick the one which is the most efficient for biofilm degradation. On the other hand, there are other three plasmids that we will add into our bacteria, which are for expressing adhesion protein, a light sensitive expression system and magnetic collection system. These specific plasmids are conducive to controlling our target bacteria.
In our project, E. coli is the bacterium carrying the function of cleaning biofilm. Some people may worry that E. coli may affect aquatic life health, but E. coli actually survive in fish body and it is a type of probiotic which is beneficial to their health . For further confirmation, we will setup experiments to test our engineered E. coli BL21 on zebrafish and look at their growth curve. Furthermore, we will control the amount of bacteria that we put in aquarium tanks according to our modelling data, aiming at achieving a significant efficiency with the smallest amount of bacteria. The magnetic-removing system would also ensure that the concentration of bacteria would be maintained in a safe level. All these works optimizes our project in a meaningful way.
E.coli-general information Retrieved from https://dnr.mo.gov/pubs/pub2401.htm