Results

We have successfully tested the function of two main systems that is important to our project. The Mec Detection System reports different intensities of red fluorescence according to different concentrations of antibiotics β-lactams, which showing excellent function for β-lactams sensing. The β-lactamase blaCMY-10 in Degradation system also shows a strong degradation ability in disc diffusion assay. For the futher design of Detective System Plus-OxySp detection system, although the experiment failed, we found the reason and determined the future direction after some investigation.

Mec detection system

1. Plasmids construction

As mentioned in our DESIGN page, Mec detective system has four parts: mecR1 (BBa_K3152000_), mecI (BBa_K3152001), mec operator (BBa_K3152002) and mCherry (BBa_J06504). To get our final functional plasmid, we constructed the following plasmids one by one to ensure that every part is working effectively. Here are three function plasmids we consecutively constructed, pSB1C3-mec operator-mCherry (Figure 1A), pSB1C3-mecI-mec operator-mCherry (Figure 1B), pSB1C3-mecR1-mecI-mec operator-mCherry (Figure 1C), respectively. In theroy, the DH5α with pSB1C3-mec operator-mCherry shows red color to the naked eyes (Figure 1A), and when there is mecI repressor protein on the plasmid, the red color disappears (Figure 1B). The third plasmid is special (Figure 1C): when there is no β-lactams, the cell has no color, but when β-lactams appears, the cell turns red (MecR1 is activated by β-lactams and degrades MecI, which active mec operator regulates transcription of mCherry).

Figure 1 All function plasmids for Mec plasmid/plasmids used for Mec detective system. (A) pSB1C3-mec operator-mCherry, (B) pSB1C3-mecI-mec operator-mCherry, (C) pSB1C3-mecR1-mecI-mec operator-mCherry.

The photos of transformants of the three function plasmids on LB agar broth are shown in Figure 2. The red bacteria colonies (Figure 2A) showed the mec operator part is working effectively. Meanwhile, the colonies of the three function plasmids from the LB solid medium had been taken and digested by EcoRI and PstI restriction enzymes to further verify the correctness of the connection. From the inserted figure of Figure 2, the results of gel electrophoresis proved the right size of each part of the plasmid. We then confirmed the results by sequencing the whole plasmids.

Figure 2 Photos of transformants of the functional plasmids on LB agar broth. (A) pSB1C3-mec operator-mCherry, (B) pSB1C3-mecI-mec operator-mCherry, (C) pSB1C3-mecR1-mecI-mec operator-mCherry. Inserted is the Gel-electrophoresis results of the plasmids after EcoRI and PstI digestion.

2. Function test for Mec Detective System

Then we tested the effect when β- lactam antibiotics existed. Ampicillin were added into 0.8 mL LB broth with a gradient final concentrations, samples were collected after culturing in 37 °C overnight. As shown in Figure 3A, we can obviously see the intensity of red fluorescence increased with the concentration of ampicilin going up before reaching inhibited concentration. The best reaction concentration is 5 μg/mL, which show the most obvious report. The consequences of Figure 3B also support the results.

Figure 3 A. Results of the RFP fluorescence intensity and OD600 under different concentrations of Ampicillin. B. Photos of E. Coli DH5α after culturing overnight under 0 and 5 μg/mL Ampicillin.

To further improve the accuracy of the Mec detection system towards β-lactam antibiotics, we tried with three different β-lactam antibiotics: ampicillin, cephalothin, cefoxitin. The culture without antibiotics was used as a control group. Figure 4 showed that with the extension of culture time, the fluorescence of mCherry in test groups gradually increased, which indicates that the expression level of mCherry in the medium gradually increased.

Figure 4 Curve of RFP fluorescence intensity and culture time under 5 μg/mL Ampicillin, Cephalothin, and Cefoxitin.

CMY-10 Degradation system

In order to find the most active and effective concentration of IPTG to induce the protein, a gradient test with the concentration of IPTG with 0.2mM, 0.4mM, 0.6mM, and 0.8mM was carried out. According to Figure 5A, it turned out that the optimum induced IPTG concentration was 0.2 mM.

The blaCMY-10 was purified by using BeyoGold™ His-tag Purification Kit. In Figure 5B, Lane 1 was loaded with protein ladder. Lane 2 and lane 3 contained the purified blaCMY-10 protein (41.9 kDa), which compared with unpurified samples showed in lane 5 and lane 6. Showing that we purified blaCMY-10 protein successfully.

Figure 5 A. IPTG concentrations gradient test for blaCMY10induction; B. Protein Gel-electrophoresis for blaCMY-10 .

Disc diffusion assay was applied to test the antibiotics performance of the blaCMY10 protein. β-lactamase was used as positive control, ddH2O was used as negative control, blaCMY10 was used as the test group. The columns were treated with three different kinds of β-lactam antibiotics (1 mg/mL ampicillin, 0.5 mg/mL cephalothin, 0.5 mg/mL cefoxitin). After 16 h incubation at 37 °C, the test group showed no visible inhibition zone around the small paper discs, indicating that blaCMY-10 effectively degraded the β-lactams (Figure 6).

Figure 6 Photos of disc diffusion assay of blaCMY10 after treating 1 mg/mL ampicillin, 0.5 mg/mL cephalothin, 0.5 mg/mL cefoxitin.

Detective System Plus-OxySp detection system

We designed a improved detective system-OxySp detection system that aiming to sense antibiotic-polluted water in larger scale. This potential funtional part is one of the detection systems for β-lactam antibiotics, which is consisted of OxyS promotor and a type of RFP, mCherry. The OxySp (a promoter of the redox-related alterations in E.coli ) inside response to antibiotics especially H2O2 that induced by antibiotics.

Figure 7 Photos of transformants of the OxySp-mCherry-OxySp-mCherry on LB agar broth. The Inserted is the Gel-electrophoresis results of the plasmids after EcoRI and PstI digestion.

We successfully constructed plasmid for OxySp detection system called “OxySp-mCherry-OxySp-mCherry”. The recombined plasmid was digested with EcoRI and PstI and checked again by sequencing (Figure 7).

However, we were a little discouraged by some difficulties during its functional test, because we couldn't trigger the production of mCherry in any way, so we went to the experts for help. After communicating with the corresponding author Jim Collins of the reference for OxySp and Professor Jim Imlay from UIUC, we have found some theoretical answers to the experimental findings in this research. Firstly, we searched for some articles again. Some bacteria species when facing lethal stressors will promote the cascade of reactive oxygen species(ROS). In some studies we have found the clues of the possibility that antimicrobials will cause the ROS cascade in bacteria cells, as one of the mechanisms of antimicrobial destruction of the bacteria cell. The OxySp promoter we found from the USE database EcoCyc, which belongs to one of the ROS cascade commonly found in the bacteria. As an upstream promoter of the gene sequence of OxyS, supposedly a small RNA gene that helps the cell to regulate and protect against spontaneous and chemically induced DNA damage. In general, OxySp promoter is triggered as the cell detects the presence of Oxidative species to protect the cell against oxidative damage^1-3.

However, we got some shocking information from experts. Prof. Imlay’s lab did not find any evidence of ROS stress when they treated E. coli with ampicillin, kanamycin, and norfloxacin. Part of their evidence was that OxyR-controlled promoters (driving synthesis of catalase, NADH peroxidase, and a protein called YaaA) (which matches our OxyS promoter) were not induced by lethal doses of these antibiotics. However, it showed that these genes were induced by authentic hydrogen peroxide as controls. In fact, another group of Jim Collins, which promoted the idea that antibiotics cause ROS, also reported virtually no induction of these same genes during antibiotic treatment. They did not present an explanation for this outcome--they presumably suspected that some other aspects of antibiotic stress were interfering with the response. In which, theoretically means, in our experiment, that ampicillin is not going to trigger the response of our OxySp promoter, not even for lethal doses. There are some published theories needed to be fixed or revised since ROS might not be one of the major causes of cell damage with antimicrobial stress. Our theory for the OxySp promoter ends here since our experiment proofs the wrong idea that we found this pathway originally, which is ROS can be detected once bacteria is under antimicrobial stress, we have to give up this pathway for real-world practice.

However, we can still utilize this pathway as another indicator of the reduced water quality if there are bacteria exists in the given water sample. We would be anticipating future works done in this related field.

References for this part investigation

1.Altuvia S , Weinstein-Fischer D , Zhang A , et al. A Small, Stable RNA Induced by Oxidative Stress: Role as a Pleiotropic Regulator and Antimutator[J]. Cell, 1997, 90(1):43-53.

2. Zhao X , Drlica K . Reactive oxygen species and the bacterial response to lethal stress[J]. Current Opinion in Microbiology, 2014, 21:1-6.

3.Altuvia S , Weinstein-Fischer D , Zhang A , et al. A Small, Stable RNA Induced by Oxidative Stress: Role as a Pleiotropic Regulator and Antimutator[J]. Cell, 1997, 90(1):43-53.

Future work

Problem solving

We have done most of our job during this iGEM season according to our project design. Also we met some problems that haven’t been solved yet. For antibiotics detection, we expecting to optimize the OxySp promoter that did not works well at present based on the advices and references we got from some relative experts and these authors we met. Moreover, we need to further test the actual wastewater to confirm that our detection and degradation system is reliable. The preliminary plan is to put the blaCMY-10 protein into the wastewater and determine the β-lactams by the color reaction of alkaline cupric tartrate solution (the solution change to blue when mixed with β-lactams).

Application

Our equipment could be divided into two cisterns: the detection cistern contains our detective biosensor and guiding polluted wastewater flow into downstream cistern for β-lactams removing, the treated water won’t be released until it up to standard. Also, We expecting that our equipment could be installed at the drainage outlet of wastewater treatment facilities, hospitals, and farms, in that case, the β-lactams wastewater will be cleaned up and remove β-lactams in time. Moreover, environmental pollution contains a variety of antibiotics like β-lactam, chloramphenicol, glycopeptide, quinolone, etc., our goal is not just to eliminate one of them. In the future, the design and construction of smart multiple antibiotics detection and degradation system are on our plan. A device that displays the concentration of different antibiotics in wastewater is needed, which is responsible to generate data reports and send them to responsible personnel or environmental protection departments quarterly.

Team:ASTWS-China/Results