Team:FJNU-China/Result
Result
- PLA
- 2-PE
- Garbage lid
- Bromidrosis
PLA
Overview: PLA is one of the core materials of our project, and it’s important to biosynthesis it. and then we find the following three key enzymes. Goal: 1. Construct three enzymes to our engineered bacteria and verify the expression of three enzymes. 2. Verify and quantitatively detect the biosynthesis of PLA. Results: 1. Realize the produce of PLA. 2. Verify the sterilization effects of PLA.
1. Three enzymes have been constructed.
We used Gibson assembly to assembly the three enzymes Tyrb, D-ldh and rocG. And the three proteins were successfully expressed. The picture below shows result of the SDS-page protein expressed by the recombinant bacteria.
Fig.1 The SDS-PAGE results of the recombinant strain: BW/pRB1s-Dldh-Tryb-rocG
2. Detected the amount of PLA by using HPLC.
Fig.2 PLA Standard curve
We firstly made samples of pure PLA with different concentration. (Use the chemicals we bought) and draw the standard curve using HPLC. Three mobile phases are double distilled water, methanol, and 0.05% formic acid in double distilled water. And the retention time of PLA is about 13.6 min.
Fig.3 PLA Chromatographic peaks
Then, dealing with our samples of bacteria, we shaking cultured 50ml bacteria liquid collected 30 OD by centrifugation and added 200ul 50mM phenylalanine as substrate, 37 degree cultured for 1h. The retention time of our sample is the same as the standard curve which prove that we realize the biosynthesis of PLA.
3. The antibacterial effect of PLA was verified.
We launched collaboration with NWU-China, TUST China and BIT-China, exploring the variety of bacteria and fungi to prove PLA’s sterilization effect. We collected their experimental data and performed statistical analysis. In this way, we knew more about the broad-spectrum antibacterial performance of PLA.TUST help us detect PLA’s sterilization effect on yeast Cen.PK2-1D.
Fig.4 Growth of yeast Cen.PK2-1D with 16 mM PLA under different OD value
Fig.5 Growth of yeast Cen.PK2-1Cwith 16 mM PLA under different OD value
Fig.6 Growth of Staphylococcus aureuswith different concentration of PLA under different OD value
Fig.7 Growth of P.aeruginosawith different concentration of PLA under different OD value
2-PE
Overview: 2-PE is another core material of our project, which has a rose like quietly elegant, delicate and persistent aroma. The content of experiment is almost same as PLA. We also found the following three key enzymes to produce it. Goal: 1. Construct three enzymes to our engineered bacteria and verify the expression of three enzymes. 2. Quantitatively detect the biosynthesis of 2-PE. Results: 1. Realize the produce of 2-PE. 2. Detect the production of 2-PE by HPLC.
1. Three enzymes have been constructed.
We used Gibson assembly to assembly the three enzymes Aro10, PAR and TyrB. And the three proteins were successfully expressed. The picture below shows result of the SDS-page protein expressed by the recombinant bacteria.
Fig.8 The SDS-PAGE results of the recombinant strain: BW/pRB1s-Aro10, PAR and TyrB
2. Detected the amount of 2-PE by using HPLC.
Fig.9 2-PE Standard curve
We firstly made samples of pure 2-PE with different concentration. (Use the chemicals we bought) and draw the standard curve using HPLC. Three mobile phases are double distilled water, Acetonitrile. And the retention time of 2-PE is about 4.8 min.
Fig.10 2-PE Chromatographic peaks
Then, dealing with our samples of bacteria, we shaking cultured 50ml bacteria liquid collected 30 OD by centrifugation and added 200ul 50 mM phenylalanine as substrate, 37 ℃ cultured for 8h. The retention time of our sample is the same as the standard curve which prove that we realize the biosynthesis of 2-PE.
Garbage lid
Overview: In the case of garbage lid, we explore the production of PLA and 2-PE further than the construction part. Moreover, the lid should express the appropriate amount PLA and 2-PE to sterilization and release fragrance.
Garbage lid-PLA
Goal: We used constitutive promoter, linked with three enzymes to express PLA. In order to make system in lid controllable, we need to figure out that how long and how much will PLA be produced. Then, go further and deduce how long the lid will start to work. Reaults: 1. Relationship between time and PLA production. 2. Relationship between cell concentration and PLA production.1. Relationship between time and PLA production.
Figure 11.The relationship between time and PLA production
We draw the line of time and different optical density (OD) of bacteria’s PLA production and found the accumulation of PLA reached 18 mM at 5 min, which means that the accumulated process of PLA is quite fast. According to the graphs, we can infer the best effecting time is 8 min for 15 OD, 6.5 min for 22.5 OD and 5 min for 30 OD.
2.Relationship between cell concentration and PLA production.
Figure 12.The relationship between cell concentration and PLA production
At the time we explored the mechanism of PLA sterilization, we found PLA inhibits the growth of harmful bacteria by break down the cell walls and membrane. It’s necessary to clarify the relationship between cell concentration and PLA production. The graph shows that the thicker engineered bacteria we use, the less PLA expressed instead. This is also a join point of our model, more details on our model page.
Garbage lid-2-PE
Goal: Because we use phenylalanine as the substrate to biosynthesis 2-PE, it’s essential to know how much substrate should be put in our lid and how long 2-PE will release into the air. Reaults: 1. Relationship between substrate depletion and 2-PE production is shown. 2. Further optimization.Relationship between substrate depletion and 2-PE production is shown.
Figure 13. 2-PE production and substrates depletion.
By controlling other variables, the line between substrate depletion and 2-PE production is shown. According to our human practice, the most comfortable concentration sets at 5 mM. More details on ourhuman practice page. We try to control the amount of substrate at 32.5 mM.
2. Further optimization.
Figure 14.Optimized 2-PE production according to our feedback from our human practice
However, the substrate of PLA is the same as 2-PE. To produce both of them, we have to add excess substrate. We came up with another idea, as is known in literature， decrease the culture temperature is a effective way to raise the production of 2-PE. After we changed the temperature, the production of 2-PE increased to 5mM, which is suitable to put in the garbage lid
Garbage lid-Suicide
Goal: Additionally, we got the LightOn switch data during our collaboration with ECUST. We planned to link it with suicide gene mazF. We try to get the result that once engineered bacteria reveal to outer environment, the toxin protein express to inhibit it. Reaults: 1. LightOn switch with mcherry 2. Verify the efficiency of toxin protein, mazF.1. LightOn switch with mcherry
ECUST helped us test the feasibility of suicide system induced by visible light. Cells with light illumination all the time, with light illumination at logarithmic phase, with light illumination at late period and cells in dark are measured fluorescence intensity. Wavelength of exciting light is 587nm, and wavelength of emitted light is 610nm.
Figure 15.The line of LightOn system linked with mCherry
The period starting light illumination doesn’t effect on the expression of mCherry, so no matter which growth phase bacteria is, the system can response.
2. Verify the efficiency of toxin protein, mazF.
Figure 16.The lethal efficiency of mazF under different time and arabinose concentration
According to the experimental results, we can know that mazF has a strong lethal effect. The death rate of E.coli connected with the concentration of arabinose. In the range of commonly used arabinose concentrations (5% to 50%), the higher the concentration, the more mazF expressed, resulting in more death of E. coli. Two hours after adding arabinose, E. coli had been efficiently inhibited.
Bromidrosis
Overview: Aim at another stink caused by bacteria, the bromidrosis is also an unpleasant smell which bothers people around. Generally speaking, we try to realize that once people sweat, PLA will express to inhibit the bacteria under axilla. Reducing the stink and give off fragrance at the same time.
Salt and temperature
Goal: In the bromidrosis part, several literatures mentioned that the salt concentration in people’s sweat is about 0.05 mM, and the temperature under the axilla is around 37 degree. We try to detect different GFP expression level by changing the salt concentration in medium and culturing temperature. Reaults: 1. Verify the salt and temperature logical gate is working. 2. Quantitatively detect the fluorescence level of circuit.1. Qualitative fluorescence level under fluorescence microscope
Figure 17.The line of time and PLA peoduction
Thus, we set 3 gradient of salt concentration, 0M, 0.05M, 0.3M, and 3 gradient of temperature, 25℃, 32.5℃ and 37℃, detected the fluorescence level took pictures under different conditions. We found the circuit is active when the environment is warmer and high osmotic. There is almost nothing under fluorescence microscope when it cultured at salt-free and 25 ℃. On the contrary, when temperature reaches 37 ℃ and salt concentration increases, GFP expression apparently increased.
2. Quantitative fluorescence level under Microplate reader
Figure 18.The line of cell concentration and PLA production
We controlled the optical density of engineered bacteria under different temperatures and did three parallel experiments to avoid inaccuracy. As the graphs showed, the circuit has apparently effect on the temperature changing, the osmotically control did have some effects but not obvious enough. The analysis of two results shows that the expression of two conditions between salt-free with 25 ℃ and 0.05M with 37 ℃ have obvious difference. We also try to change GFP into PLA and get further data to analysis the circuits, but limited by time.
tetR and tetO
Goal: The Tet-Off system has been applied to several organisms, which turns expression of its controlling genes OFF. Base on the metabolic state switch, we have switched between production mode and suicide mode. We characterize the working effect of our circuits by the fluorescence expression levels of GFP and RFP. Reaults: 1. Determine the optimum content of the inducer. 2. Quantitatively detect the fluorescence expression level. 3. Verify circuit implementation mode switching.1. In the production of 2-PE mode, by adding an appropriate amount of inducer (anhydrotetracycline, ATc) to the lotion, PLtetO1 initiates expression of the downstream product, and the red fluorescent expression effect is observed under a fluorescence microscope.
Figure 19. Fluorescence levels per OD at different inducer (anhydrotetracycline, ATc).
Figure 20.The line of cell concentration and PLA production
We set five different inducer concentration 10 ng/ml, 50 ng/ml, 80 ng/ml, 120 ng/ml, 150 ng/ml (anhydrotetracycline, ATc). Fig. 20 illustrated that the measured RFP fluorescence value increased from ATc concentration of 10 ng/ml to 80 ng/ml. The red fluorescence at different ATc concentrations can be visually observed, and it is concluded that the expression of red fluorescent protein was indeed controlled by the PLtetO1 promoter as the ATc concentration was less than 80 ng/ml. At a ATc concentration of 120 ng/ml to 150 ng/ml, the fluorescence intensity value was significantly weakened. We speculated that higher ATc concentrations (more than 120 ng/ml) had an adverse effect on cell growth.
In further experiments, we set up a control group (CMV promoter initiation) and found that excessive addition of ATc would affect the expression of fluorescent protein and inhibit the growth of the strain. We are looking for the most suitable addition to ATc, which will guide our product design.
2. In the suicide mode, safety is ensured by expressing the toxin protein mazF. The arabinose was added, PBAD initiated the expression of the downstream product, and the green fluorescent expression effect was observed under a fluorescence microscope.
Figure 21. Fluorescence images at different arabinose concentrations
We set 3 level arabinose concentration (higher=50%, medium=25%, none=0%) Fig. 21 illustrated that the measured GFP fluorescence value increased by the addition of arabinose. The green fluorescence at different level arabinose concentration can be visually observed, and it is concluded that the expression of green fluorescent protein was indeed controlled by the PBAD promoter .
3. Using bacteriostatic experiments, we obtained more accurate experimental data
Figure 22. Reflects the lethal state of the toxin protein
Fig. 21 Under the condition of adding arabinose concentration of 50% and reaction time of 150 min, we can achieve the best lethal efficiency. According to our human practice, the most comfortable concentration sets at 5 mM. (more details on our human practice) We try to control the amount of substrate at 32.5 mM. However, the substrate of PLA is the same as 2-PE. To produce both of them, we have to add excess substrate. We came up with another idea, changing the RBS to increase the production of 2-PE. After we changed it to a strong RBS, the production of 2-PE increased to 5mM, which is suitable to put in the garbage lid. More details can be found on our Model page.