Team:Tsinghua/Results
Result
NOS cloning and NO detection
NOS cloning
In our experiment, we first cloned NOS gene in Bacillus subtilis by PCR (shown as Fig.1) and attached it to Pet28-a for determination using double restriction enzyme and DNA ligase. Then the ligated vector was transformed into the BL21(DE3) for protein expression and DH5α for plasmid application. The plasmid was sequencing by company, which was accord with our design.
Figure1. The result of NOS PCR from B.subtilis genome
Then we do the Western blot to make sure our system could express protein. The plasmid PET28-a itself contains a his-tag sequence, which is attached to the final of NOS sequence. So we can use antibody which can detect his-tag to detect the protein. To induce the production of target protein, the overnight culture at 37℃ was prepared. Arginine was added to 40ml LB(containing 100μg/ml kanamycin) to final 0.5mM concentration. ALA was added to final 0.45mM concentration and 400μl microbial was added. Then Incubate at 37℃ until OD600=0.5. Absorb half of the liquid and add it to the new conical flask as control. IPTG was added to the induced group to final 0.5mM concentration. All the groups were incubate at 28℃.The expression of 0-4h was detected. At the same time, GFP-His was added as the positive control.
Figure2. The result of western blot. The expression level of NOS-HIS gradually increased with the change of time.
NO detection
we separately measured the change of NO over time in the NOS transformed BL21 (DE3) culture. The change of nitrite in the bacterial solution without IPTG induction with time was measured with total NO detection kit (Beyotime). We found that the nitrite content in the bacterial solution also decreased at the initial stage. This may be caused by the consumption of nitrite by the growth of bacteria, so we want to reflect the production of NO by the difference value of nitrite content between IPTG induced（IPTG+）and no IPTG induced（IPTG-）. The results are as followed.
Figure.3 The difference in NO production by time
C4-HSL quorum sensing
As our expectation, the engineered bacteria could sense the C4-HSL signal derived from P . aeruginosa. Thus, a C4-HSL report system should be considered and established. Here we characterized the composite part BBa_K1893003 as the report system. Since this part was not distributed, it was synthesized in BGI QINGLAN BIOTECH according to the sequence offered on the website and then integrated into plasmid pSB1C3. (The backbone of pSB1C3 is derived from distributed part BBa_R0071)
Note: the Rhl and pRhl are WT and not improved with relatively lower expression level, compared with the part improved by iGEM 2014 Tokyo_Tech.
Here we tested the working condition of 3 plasmids in E . Coli strain BL-21(DE3). The tested plasmid, pSYS, shares the same sequence with BBa_K1893003. For the other plasmids, pPRhl is derived from part BBa_R0071 and plasmid pSYS(LVA) is modified from pSYS (in which the Rhl protein is labeled with LVA tag), which serve as the control groups.
Note: LVA tag is used as degradation signal for cellular protein.
After transformation, the single colony was picked and inoculated into 2mL chloramphenicol LB to culture overnight at 37℃. Then 60 times diluted of the culture solution was prepared. After 60-90min of 220 rpm shake at 37℃, the OD600 of the solution reached 0.3-0.5. Then solution was separated into 3 groups (500 μL) and meanwhile different inducers (0.5 μL) were added. After 4 hours incubation at 37℃, the cell culture was aliguoted into 96-well plate, 100 μL per well, 4 wells per group. VARIOSCAN FLASH(TM) microplate reader was used to measure the RFU(Ex 490 nm, Em 525 nm). The value of each column(RFU/Turbidity) was calculated by(RFU/OD600).
As the data illustrated below, the C4-HSL report system pSYS shows about 9-fold signal enhancement with 100μM C4-HSL and about 6-fold enhancement with 10μM C4-HSL(shown as Figure A). Another experiment was used to quantify the concentration of C4-HSL and the RFU(shown as Figure B).
Figure.4A Test of C4-HSL report system in BL-21. Plasmid were transformed into BL-21 and the value of RFU/Turibidity was measured. The C4-HSL report system pSYS shows about 9-fold signal enhancement with 100μM C4-HSL and about 6-fold enhancement with 10μM C4-HSL.
Figure.4B RFU/turbidity in C4-HSL gradient tested in BL-21. It is expected that this report system is able to response well to more than the threshold of 1μM C4-HSL
Test of C4-HSL report system in different strains
Here we also tested the plasmid pSYS in other E . Coli strains including DH5ɑ and TOP-10, (shown as Figure C) indicating that the C4-HSL report system may have the highest efficiency in TOP-10 strain.(about 19 fold enhancement with 100μM C4-HSL and 13 fold enhancement with 10μM C4-HSL) The working condition of the report system in DH5ɑ is similar to the case in BL-21.
Figure.5 Test of C4-HSL report system in different strains. Each strains were tested in 3 parallel group with different treatment. The C4-HSL report system seems to have highest efficiency in TOP-10 strain and relatively lower efficiency in BL-21 and DH5ɑ.
Detection of C4-HSL concentration in P.aeruginosa biofilm
Figure.6 C4-HSL produced by PAO1 biofilm is detected by QTRAP Liquid chromatography-mass spectromer .
We detected the C4-HSL produced by PAO1 biofilms by QTRAP Liquid chromatography-mass spectromer. (shown as figure 6)The detected range of the biofilm produced C4-HSL concentration will be used as a reference for QS system inducing (using C4-HSL to induce the constructed RhlR system to produce fluorescence signal.
Biofilm culture and quantification
Biofilm Degradation Using SNP
Degradation analysis of crystal violet staining
We used chemical donors of NO—Sodium Nitroprusside (SNP) to verified the effectiveness of NO in biofilm degradation and determine the degradation threshold of NO for our engineered bacteria. Degradation experiments of P.aeruginosa biofilms with different concentrations of NO were firstly performed on the 24h-grown PAO1-GFP biofilm with GFP signal and measured after 24h.
After investigation, we used the ratio of biofilm (in situ crystal violet staining absorbance at 540nm) to the biomass of planktonic bacteria (fluorescence signal of GFP 488nm in supernatant) to reflect the degradation of biofilm. From the column chart, we can see the SNP has degradation effect on the biofilm, especially from the 200μg/mL to 200ng/mL level (Figure 7). The high ratio of biofilm/planktonic biomass at high SNP concentration is caused by the planktonic bacteria killed by the high-concentrated NO. By measuring the degradation of the biofilm at different SNP concentrations, we determined the optimal concentration range of SNP that could degrade the biofilm, which gives implication for the detection of NOS expression.
Figure 7. The biofilm degradation using SNP measured by the ratio of biofilm/planktonic biomass, SNP has strong degradation effect at 200μg/mL to 200ng/mL
Visualisation of SNP degradation
To further confirm the degradation effect of SNP, we use Nikon A1R HD25 confocal laser scanning microscope to obtain the 3D time-lapse images of biofilm degradation. The biofilm is imaged by the GFP fluorescence signal of PAO1-GFP. And we select the SNP concentration of 50μg/mL to degrade the biofim. Biofilm 3D time-lapse movies were obtained and cropped using NIS Elements AR software.
In the movie of 5h degradation, we can see that the green fluorescence of the largest group of biofilms in the middle is significantly reduced, and the fluorescence of the surrounding small biofilms also gradually disappears, and the whole biofilm is continuously degraded and dispersed (Figure 8). So we visualize the biofilm degradation effect of NO with the movie.
Figure 8. The biofilm degradation using SNP visualized by 3D time-lapse imaging
Coculture of E.coli and P.aeruginosa
In order to test whether E.coli and P. aeruginosa can coexist, the stability of our engineered bacteria in the actual bacterial membrane environment can be verified. If our engineered bacteria were to be competitive and eliminated in a short time by the rapidly growing P.aeruginosa, then our engineered bacteria might not survive in the actual biofilm and continue to release NO. We hoped to observe the ecological dynamics of E.coli in p. aeruginosa biofilm through coculture experiment, and inoculated E.coli at different times using confocal imaging. P.aeruginosa biofilms were imaged and analyzed.
An E. coli BL21-mCherry strain inserted with plasmid pUC encoding mCherry fluorescent proteins was used to study dual-species biofilm growth behavior. This strain is derived from E. coli K-12. A P. aeruginosa PAO1-GFP strain with a chromosomally expressed green fluorescent protein (GFP) was also used. P. aeruginosa was imaged by GFP fluorescence, while E.coli was imaged by constitutively expressed mCherry fluorescence.
In the 72-hour coculture experiment, we observed that BL21-Mcherry could stably exist and grow in Biofilm. We calculated the ratio of mCherry and GFP signal strength in confocal image to reflect the dynamic changes in coculture experiment under the circumstances of inoculation of E.coli at different times. We found that E. coli had the best growth status in P. aeruginosa biofilm when inoculated with E.coli between 8 and 16 hours (Figure 9&10). Our co-ulture experiment proved that E.coli can grow in P. aeruginosa and will not be excluded in a short time. This provides some evidence that our engineered bacteria can stably express NO in the biofilm.
Figure 9. The planar and 3D image of coculture of E.coli and P.aeruginosa with E.coli inoculation at 0h and 16h
Figure 10. The coculture dynamic indicated by the intensity proportion of E.coli and P.aeruginosa with E.coli with different time of inoculation
Suicide Switch
Construct the kill switch controlled by eLightOn system
Because of the COVID-19 epidemic, we just started experiments with the kill switch in late September. So we have not completed the construction of the kill switch. We designed the method to construct the kill switch controlled by eLightOn system and will finish it in the future. According to the literature related to the eLightOn system, we will synthesize a fragment of DNA containing LexRO gene, constituent promoter ColE and cognate operator LexA408. We will obtain the fragment containing miniColicin by PCR from the plasmid containing miniColicin. We will insert two fragments into a plasmid using the Golden gate Assembly method and then transform the recombinant plasmid into Escherichia coli DH5α. Positive clones will be screened by solid medium plates coated with corresponding antibiotics and sequencing. The recombinant plasmid is shown as Figure C.
Figure C. The map of recombinant plasmid containing kill switch controlled by eLightOn system drawn by snapgene.
Test the kill switch controlled by eLightOn system
Because of the COVID-19 epidemic, we just started experiments with the kill switch in late September. So we have not completed the test of the kill switch. We have designed an experiment to test the performance of the kill switch. We will inject Escherichia coli DH5α containing kill switch plasmid into 3.05mL liquid LB medium with corresponding antibiotics and then cultured it to stationary phase. Then we will take 50μL liquid LB medium to spread the plate and then estimate the population of bacteria by CFU. Then we will divide 3mL liquid LB medium into 3 tubes for culture. The first tube will be cultured in darkness as blank control. The second tube will be cultured in blue light of 460nm and 30W/cm2, which can trigger the light-controlled kill switch. The last tube will be cultured in natural light as we want engineered bacteria to trigger the light-controlled kill switch under nature conditions so that we don’t need add blue light at the end of the tube to trigger the engineered bacteria to suicide. Take out the liquid LB medium to spread the plate at regular time intervals to determine CFU. Estimate the population of bacteria by CFU. Compare the changes in bacterial populations to test the performance of the kill switch under blue light and under natural light.
The miniColicin derives form BBa_K1976048.
Note: If the population of bacteria is too high to count the CFU, dilute the liquid LB medium containing Escherichia coli DH5α.
Reference:
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[2]Gusarov I, Starodubtseva M, Wang Z Q, et al. Bacterial nitric-oxide synthases operate without a dedicated redox partner[J]. Journal of Biological Chemistry, 2008, 283(19): 13140-13147.
[3]http://parts.igem.org/Part:BBa_K1893003
[4]Barraud N. Nitric oxide-mediated differentiation and dispersal in bacterial biofilms[J]. School of Biotechnology and Biomolecular Sciences, 2007.
[5]Gui M, Liu L, Wu R, et al. Detection of new quorum sensing N-acyl homoserine lactones from Aeromonas veronii[J]. Frontiers in microbiology, 2018, 9: 1712.
[6]Culotti A, Packman A I. Pseudomonas aeruginosa promotes Escherichia coli biofilm formation in nutrient-limited medium[J]. PLoS One, 2014, 9(9): e107186.
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