Team:Nanjing-China/project

Overall Project

Atrazine is one of the most heavily used herbicides worldwide, where it is used to control the growth of weeds. Atrazine is also a persistent environmental pollutant, and widespread contamination of groundwater has been reported in the United States. As such, there are increasing concerns over the toxicity of atazine in the environment.
Considering the fact that atrazine can hardly be degraded naturally in soil and water, we design and construct a system in E.coli to make them move torwards atrazine simultaneously up to the gradient and absorb it, degrade it into a harmless chemical substance.
At the same time, we also use the computer calculation to simulate the system we construct, hoping to endow our system with high stability.

Pre-experiment

　　　Through consulting literatures and materials, we found that the concentration of atrazine used in experiments related to its degradation were the most changeful. As we all know, atrazine, which is toxic to bacteria, can reduce the amount of bacteria, and influence the efficiency of degradation. To avoid this problem as well as to provide the public with a standard of atrazine concentration in experiments, we conducted a series of pre-experiments, studying the relationship between atrazine and bacteria.
Experiments and Results
　　　The bacteria strain we used is called K-12, a strain isolated from Escherichia coli. It is a type strain which moves the fastest in soil of all strains with clear genetic background and handy genetic modification.
　　　As Fig. 3-2-1 shows, when the concentration of atrazine was between 200μM and 500μM, the trend of the growth curve was similar to the nature growth curve without any atrazine. However, when bacteria exposed to atrazine of 1000μM, the growth curve of K-12 had changed a lot, especially in plateau period.



　　Growth Curve of K-12 cultured in different concentration of atrazine. Atrazine could not restrain bacteria growth significantly if the concentration was between 200μM and 500μM, but the growth curve changed a lot when the concentration of atrazine rose to 1000μM, suggesting that high concentration of atrazine is harmful to K-12.
　　　In order to make their relationship more intuitive, we performed another experiment, comparing the amount of bacteria in plateau period. As illustrated in Fig. 3-2-2, the amount of bacteria cultured in atrazine of 200μM or 500μM hardly changed, while it declined sharply when cultured in concentration of 1000μM.
　　　Fig.3-2-2 The amount of bacteria cultured for 10h in different concentration of atrazine. After 10h, plateau period had been reached. It was obvious that the maximum of bacteria exposed to atrazine of 200μM or 500μM hardly changed, but it became much lower when the concentration turned to 1000μM.


　　　Taking the condition of bacteria's growth for consideration, we finally chose 200μM-500μM as the concentration of atrazine in our all experiments.

Transporter

Currently, Pseudomonas sp. strain ADP seems to be the optimal bacteria strain for atrazine degradations, which appears to be the sole nitrogen source for the bacteria. A study has found that a large plasmid carries all the genes and complex pathways related to atrazine degradation in Pseudomonas sp. strain ADP. We analyzed all the genes in it and finally found a multiple transmembrane protein which we called TRM (Fig. 3-3-1). We hypothesized that the function of transmembrane protein was related to atrazine transportation and thus confirmed it with several experiments.



Fig. 3-3-1 TMHMM posterior probabilities for WEBSEQUENCE. TRM has 11 trans-membrane domains. In prokaryotes, a protein like this has been considered as a potential transmembrane protein.

Experiments and Results
　　　To prove that TRM is a transmembrane protein, we constructed a circuit where a constant promoter drives the expression of the TRM which is fused with a green fluorescent protein to localize it. And the result of transmembrane protein verification is shown in Fig. 3-3-2, suggesting that TRM is located on the cell membrane.



　　　Fig. 3-3-2 The circuit we constructed to verify the function of TRM and subcellular localization of TRM-GFP fusion protein. (A) The circuit we constructed to verify the function of TRM. (B) Fluorescent microscopy image of cells expressing TRM-GFP (green) shows that TRM distributed throughout the cell membrane.
　　　To prove that TRM is related to transportation of atrazine, we used HPLC to analyze the concentration of atrazine after incubating engineered bacteria in 500μM atrazine for 24h (Fig. 3-3-3).It is obvious that bacteria with TRM can reduce the concentration of atrazine, suggesting that TRM plays an important role in transportation of atrazine.



　　　Fig. 3-3-3 Atrazine concentration of K-12 with or without TRM. "0h" stands for control group with no atrazine and the bacteria have been incubated for 0 hour. "Wild Type" stands for Escherichia coli K-12 which ispositive control and "Vector" stands for K-12 which contains a plasmid-pGFP which is negative control. "TRM+" is the experimental group where a plasmid-pGFP which carries TRM was transformed into Escherichia coli K-12. And the result shows that K12 is significantly different from TRM+ in the statistical sense and it also indicates that TRM plays an important role in transportation of atrazine.

QS System

First of all, we should have an understanding of how bacteria move. For E.coli, they have flagellar motor to control their movement. The direction of rotation of it is controlled by the protein CheY. WhenCheY is not phosphorylated, the flagellar motor rotates counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. PhosphataseCheZ, dephosphorylates CheY-P and causesthe flagellum to rotate CCW. E. coli lacking thecheZ gene (ΔcheZ) cannot dephosphorylateCheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control over gene cheZ to control the motility of our bacteria.
Fig.0

　　　We would like our bacteria able to seek and destroy atrazine in the soil. To achieve this goal, a classic systemcan do the job. In this system, bacteria could move around randomly but fixed when sensing atrazine as the ribosome-switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assembly around the target molecules after a period of time.
Fig.1 (gene circuit of Pattern 1)

　　　However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it.For better control over the function of our bacteria, we take the quorum sensing system into consideration. We expect some bacteria which first find the atrazine would recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuit to realize it.



Fig.2 (gene circuit of Pattern 2)
　　　This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopt the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone(AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR.Our atrazine-sensing module control the translation of LuxI through riboswitch which turns on when combined to atrazine. As for the recruiting module, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. The higher cell density is, the higher AHL concentration is, and the stronger transcription of cheZ which means greater motilityis, leading to directional movement towards bacteria which send out the signal.Finally, a repressor CI, controlled by riboswitch which turns on when combined to atrazine,represses the promoter Plux/CI and serves as a brake of the movement, resulting in bacteria fixation at source of pollution.
　　　We establish mathematical models and use computer simulation to compare these two systemsand evaluate the improvement. In this case, we design the third system, which has the clearest logical relations, to help us better understand how the gene circuits work.
Fig.3 (gene circuit of Pattern 3)
The atrazine-sensing module is the same as the one in Pattern 2. However, the double-functional promoter Plac/CI is replaced by a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Plambda, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by riboswitch, expresses to brake.
Method
1.Verification of gene cheZ in genetic level
2.Verification of gene cheZ from phenotype
3.Verification of lux system

Ribosome Switch

Ribosome switch is used widely in synthetic biology to detect various small molecules. This summer, we chose an efficient ribosome switch which can detect atrazine created via a new method (NAT CHEM BIOL, Joy Sinha. et al., 2010). And then, our team verified the basic parameters about this amazing ribosome switch, including the lowest detect concentration and the induced time.

Experiments and Results
Our team used this kind of ribosome switch which can detect atrazine to control the GFP expression in E.coli. E.coli expressed GFP after 20 hours under 500μM of atrazine, but could not express GFP without atrazine (Fig. 3-4-1). This shows the ribosome switch can be turned on under 500μM of atrazine.



　　　Fig. 3-4-1 Ribosome switch used in our experiments can detect atrazine. (A) The principle of how the ribosome switch works. (B) Observation of GFP under confocal microscope. Ribosome switch can be induced by atrazine under 500μM of atrazine.
　　Our team used this part to control GFP expression in E.coli. We found all E.coli had expressed GFP under all the concentrations of atrazine, from 1μM to 500μM, in our experiment (Fig. 3-4-2). This data indicates that the ribosome switch can work under 1μM of atrazine.
　　　Fig. 3-4-2 Various concentrations of atrazine were detected after 20h culturing.



　　Our team used this part to control the GFP expression in E.coli. We found E.coli had merely expressed GFP after 12 hours under 200uM of atrazine. This data elucidates that the ribosome switch can work after 12 hours under 200uM of atrazine.
Fig. 3-4-3 The ribosome switch was induced at different time under 200uM of atrazine.



According to these data, this ribosome switch is more efficient than the one used before. Therefore, we are confident of the fact that we can put this amazing part into detecting atrazine in real life.

Degrading Enzyme

　　　Atrazine is quite hard to degrade naturally in the soil, which makes it persist for a long time in the environment once used. This phenomenon can be considerably severe in that atrazine would cause metabolic disorders both in animals and humankind. For many years, we didn't have an efficient solution to this problem.
　　　However, the discovery of the super power of Arthrobacter aurescens provides us with a light of hope. This amazing species is capable of utilizing atrazine as a sole source of carbon with the help of a series of degrading enzymes which can metabolize atrazine into a kind of nontoxic substance. In our project, we utilize the most useful degrading enzyme TrzN to degrade atrazine. We even mutated the TrzN to make it degrade faster. At the same time, we found a transmembrane transporter TRM, which involves in transporting atrazine from the outside to the inside of the bacteria so that atrazine can be better degraded.

Experiments and Results
　　　We took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group TrzN- and Group TrzN+, 3 tubes each. With the same protocol used to test the function of TRM, we found that TrzN could distinctively degrade atrazine with the bacteria cultivated 24 hours in 37℃ (Fig. 3-6-1). The difference between wild type and TrzN+ was distinctive, suggesting that TrzN is a degrading enzyme of atrazine.


　　　Fig. 3-6-1 TrzN can distinctively degrade atrazine. This figure shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Group 0 means no bacteria in the cultures; cultures in Group Wild Type contained the wild type K12 bacteria; cultures in Group Vector contained the strain K12 with pGFP; and cultures in Group TrzN+ contained the bacteria which are able to express TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can conclude that TrzNis an efficient degrading enzyme in solving the problem of TrzN.
　　　We also combined the TRM and TrzN together can make the degradation more efficient (more information about TRM. This time, we combined the TRM and TrzN together, under the same method of testing the function of TRM and TrzN, we found that the combination of TRM and TrzN could decrease the atrazine concentration in the bacteria culture to a greater extent (Fig. 3-6-2).

　　　Fig. 3-6-2 Combining TRM and TrzN together can make the degradation more efficient. shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Apart from groups shown above, cultures in Group TRM+TrzN+ contained the bacteria which are able to express TRM as well as TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can see that the atrazine concentration in supernatant of Group TRM+TrzN+ was even distinctively less than that in supernatant of TRM+. This means combining TRM and TrzN is indeed a wise choice to polish up our system.
　　　According to these data, we find that TRM can indeed transport atrazine from the outside to the inside of the bacteria, and of course, TrzN can degrade atrazine well and fast. Therefore, we could be confident of the fact that we may one day utilize our system to solve the problem of atrazine in real life.