Team:NEU CHINA/Demonstrate

 WET LAB_R&D

     In order to simulate the inflammatory NO, 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used to continuously release NO and the final concentration was stable at about 5.5μM, which was the same as the NO concentration in IBD patients [1]. We used 100 μM SNP solutions for NO sensor sensitivity testing.
    For the NO sensor sensitivity testing, we transformed the constructed plasmid with NO sensor into E. coli BL21 competent cells. Competent cells were cultured at 37 ℃ overnight, and then diluted to $OD_{600}$ = 0.4. And then, cultured bacteria at 37 ℃ for 1.5 hours, the appropriate concentration of 100μM of inducer SNP aqueous solution were added. After 2 hours of SNP induction, we detected the expression of the luciferase by Luciferase assay (from Beyotime RG005). The Luminescence data indicated that the NO released by the SNP aqueous solution could effectively activate the expression of the reporter gene. (Fig. 2)

WET LAB
    However, the results also demonstrated the serious leakage problem which was the same as last year. So, we inserted an extra NsrR binding sequence (NsrRBS) downstream of PyeaR to create a ‘roadblocking’ effect [3] (Fig. 4A). Comparing to the unmodified PyeaR-luc system (Fig. 4B), the histogram of luminescence data demonstrated that the relative lower luciferase signal in PyeaR-NsrRBS system in the absence of NO.
Figure 6. Diagram for fixed-gain amplifier in pCDFDuet-1. T7 promoter is responsible for downstream gene transcription under the IPTG activation. The ribosome binding sites (RBSs) were indicated as red color. The hrpR and hrpS are activator proteins’ coding genes, PhrpL, a promoter which can be induced by the ultrasensitive high-order co-complex HrpRS. In this system, we used Green Fluorescent Protein (GFP) as our reporter protein. pCDFDuet-1, the plasmid for expression with streptomycin resistance.
    To find and show a clear result of tunable amplifier, T7 promoter would be replaced by the constitutive promoter J23109. Unfortunately, we have not constructed this part with the pCDFDuet-1 plasmid during this iGEM year. Our experiment has stalled in the extraction of plasmid DNA, even attempting too many times. After communicating with our adviser and consulting with the synthetic companies, the plasmid might not be suitable for such a part. We tried to construct it on other plasmids. But for the time limitation, we built the Tuner Model to modify the tunable-amplifier in our “gut firemen” instead. (details about the Model, please click here).
1. Nitric oxide sensor
RESULT & DEMONSTRATE
    For details about our project description and inspiration , please click Description & Inspiration.
 
    For details of whole project design, please click Design.
 
    As a therapeutic project, we are aware of the difficulties to verify the entire system in less than one year. We split the entire system into four core parts and verified them separately: the nitric oxide sensor (nitric oxide, NO, is a biomarker of intestinal inflammation); “amplifier & tuner” the bio-amplifier; anti-inflammatory proteins production and the “kill switch” system construction. We believed that the successes of these core parts will ultimately demonstrate the entire system functioning in alleviating IBD symptoms.

    Afterwards, we also transformed the PyeaR based NO sensor in our chassis, E. coli Nissle 1917 (EcN). The results were consistent with our expectation which confirmed the precise colonization of the engineered EcN in the inflammatory region (Fig. 3).
Figure 3. The response to NO of PyeaR-luc in E. coli Nissle 1917. Histogram of Luminescence (RLU): The luminescence signal can be detected from the empty vector and the PyeaR-luc vector under either SNP induction or not.
    To verify the fixed-gain amplification capability in an apparent way (Fig. 6). At first, we integrated the T7 promoter as the nitric oxide responsive transcriptional sensor to the input of the fixed-gain amplifier with GFP as the output.
2. Amplifier & Tuner
Figure 7. Responses of the GFP without fixed-gain amplification (V-GFP) and with fixed-gain amplification (V-AM). The cells are induced by 5 varying concentrations of IPTG (0, $10^{-6}$ M, $10^{-5}$ M, $10^{-4}$ M, $10^{-3}$ M) after 4 and 6 hours (A, 4h; B, 6h).
    In order to detect the NO specifically and accurately, firstly, we constructed a yeaR based NO sensor in pCDFDuet-1 plasmid, which also used by previous teams’ work. When NO bounds to NsrR (the PyeaR’s transcription inhibitor), halting the repression and allowing the expression of lucifersase. To test the NO sensors’ sensitivity and specificity, we chose luciferase as our reporter gene, because of the extremely sensitivity allows quantification in even small changes in transcription (Fig. 1).   
Figure 4. Design and characterization of the improved NO sensors. A. Diagram for improved NO sensor system in pCDFDuet-1. PyeaR, a promoter which is sensitive to NO. Native NsrRBS, the native NsrR binding sequence. Extra NsrRBS, the extra NsrR binding sequence. Luciferase, reporter gene. Terminator B0010/B0012, double terminator. B. Comparison genetic leakage expression of PyeaR-Luc and PyeaR-NsrRBS-Luc systems with or without NO induction. Blue bars indicate the luciferase expression percent under the NO induction, while Red bars show the percentage of genetic leakage without NO induction.

    After that, based on Saraiva’s team’s work, the ytfEΔ strain is much more sensitive to nitric oxide than the parental E. coli strain [2]. Thus, we constructed a novel sensing system PytfE-Luc to compare with our previous sensors to demonstrate the most sensitive and specific NO sensor (Fig. 5A). Thus, the results demonstrated that the ytfE based NO sensor’s output was nearly five times higher than the yeaR based NO sensor’s (Fig. 5B), which convinced us that the novel NO sensor, PytfE is more sensitive.
    We transformed 3 different vectors into E. coli BL21 competent cells. Amplifier and GFP sequences were inserted into pcdfDuet-1 vectors which were called V-AM and V-GFP. Empty vectors with amplifier or GFP were named VE, VE-GFP and VE-AM. After vector transformation, we grow the transformed competent cells on the LB plates with streptomycin, once we got colonies, we cultured single colony in 5 ml LB medium overnight at 37 ℃. Next day, we washed bacteria by 1ml PBS and measure the cell growth pattern by photocytometer. In order to read the fluorescent signals, we used LB-S (LB with streptomycin) to dilute bacteria and added them into the black 96-well plate (Cat#: 3916, Corning Corp.) with $OD_{600}$=0.025 per well. Five different Isopropyl-beta-D-thiogalactopyranoside (IPTG) concentrations (0,$10^{-2}$ M, $10^{-3}$ M, $10^{-4}$ M, $10^{-5}$ M and $10^{-6}$ M)  were used to induce the T7 promoter activation for 4 or 6 hours. After IPTG induction, the Enzyme Labeling Instrument were used to detect the green fluorescent signal. The excitation wavelength was 485 nm with the absorption wavelength 525 nm.
    When the transduced transcriptional input from the T7 promoter was connected to our amplifier, the resulting output signal amplitude and dynamic range increased significantly as well as the response sensitivity to the inducer (Fig. 7).

Figure 8. Diagram for tunable-gain amplifier in pCDFDuet-1. As similar to fixed-gain amplifier in pCDF-duet1 plasmid, this system included PBAD promoter that was enabled to activate hrpV transcription and inhibit the hrpS effector protein function.
    For the reducing regulation when the inflammation was not so serious, we designed another tunable-gain amplifier with the inhibitor protein, HrpV(Fig. 8).
Figure 2. The response to NO of PyeaR-luc in E. coli BL21 competent cells. Histogram of Luminescence (RLU): The luminescence signal can be detected from the empty vector and the PyeaR-luc vector under either SNP induction or not.
Figure 5. Design and characterization of the ytfE based NO sensors. A. Diagram for ytfE based NO sensor system in pCDFDuet-1. PytfE, a promoter which is sensitive to NO. Luciferase, reporter gene. Terminator B0010/B0012, double terminator. B. Comparison genetic expression of PytfE-Luc and PyeaR-Luc systems with or without NO induction. Red bars indicated the PytfE’s luciferase expression percent under the NO induction. Blue bars indicated the PyeaR’s luciferase expression percent under the NO induction. Yellow bars showed the percentage of PyeaR’s leakage without NO induction.

REFERENCE

[1]Ljung, T., et al., Rectal nitric oxide assessment in children with Crohn disease and ulcerative colitis. Indicator of ileocaecal and colorectal affection. Scand J Gastroenterol, 2001. 36(10): p. 1073-6.
[2]Justino, M.C., et al., Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbiol Lett, 2006. 257(2): p. 278-84.
[3]Fernandez-Rodriguez, J. & Voigt, C. A. Post-translational control of gencircuits using Potyvirus proteases. Nucleic Acids Res,2016. 44(9) :6493–502

Amplifier
Tuner
3. Production
    In our design, we have come up with a thinking of expressing the immune cytokine, IL-10 and the enzyme, myrosinase, and bringing them out of cells with secretory tag YebF. It has been reported that these two proteins can be expressed by bacteria with biological activity. Since we did not have access to the sequence used in the last year NEU_China_A team’s plasmid, we demonstrated the characterization directly to verify the plasmids from last year’s team. After that, we were able to test the therapeutic compounds’ activities.
Myrosinase
Figure 9. Diagram for the myrosinase’s coding gene in plasmid, pcold-1. Promoter PcspA, a super strong promoter when incubating at low temperature. RBS, the ribosome binding site. Myrosinase, the coding gene of Myrosinase.
    The gene circuit had been constructed on pcold-1 plasmid (Fig. 9) and the immunoblotting demonstrated the expression of myrosinase. The gene myrosinase inserted in pcold-1 vector was transformed into E. coli BL21 strain. After incubating at 37 ℃ for 12h, CFU was inoculated to LB medium followed by 12h’s incubation. The culture was then diluted to $OD_{600}$=0.2 and re-cultured to $OD_{600}$=0.5. Then, 1 mM IPTG was added to the culture media to induce protein expression followed by 12h incubation. After induction, bacterial cells were washed and collected, prepared for immunoblotting. The expected molecular weight of protein myrosinase was 57.46 kDa (Fig. 10).
Interlukin-10
Figure 11. Diagram for mouse IL-10 expressing and secreting in pCDFDuet-1. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. RBS, ribosome binding site. Secretory tag YebF is introduced to secret protein downstream.
    The expression vector has been constructed using pCDFDuet-1 (Fig. 11) and immunoblotting was used to express the IL-10. Expressing vector harboring IL-10 gene is transformed into E. coli BL21 strain. After incubating at 37℃  for 12h, CFU is inoculated to LB medium followed by 12h incubation. The culture was then diluted to $OD_{600}$=0.2 and re-cultured to $OD_{600}$=0.5. Then, 1 mM IPTG is added to the culture to induce protein expression followed by 12h incubation. After induction, bacterial cells were washed and collected, prepared for immunoblotting. The expected molecular weight of protein YebF-mIL-10 was 33.46 kDa (Fig. 12).
Figure 12. Protein expression of mouse IL-10 gene in E. coli BL21 strain. After induction of IPTG at final concentration of 1mM, the culture was incubated at 37℃ for 12h followed by immunoblotting.
Secretory tag
Figure 13. Diagram for human IL-10 expressing and secreting in pCDFDuet-1. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. RBS, ribosome binding site. Secretory tag YebF is introduced to secret protein downstream.
Protocol
1. Incubate the inoculum at 37℃ overnight.
2. Dilute the culture to $OD_{600}$=0.2, followed by incubation at 37℃ for around 2 hours until the $OD_{600}$=0.6.
3. Add IPTG at final concentration of 1mM.
4. Incubate the culture overnight.
5. Remove cells by centrifugation at 5000rpm for 10min.
6. Remove cells using filter.
7. Add saturated ammonia sulfate to the supernatant, and then agitate softly for 2 hours while sitting on the ice.
8. Span the protein down by centrifugation at 13000rpm for 30min.
9. Resuspend the protein by cold water and remove any faults by filter.
10. Add Loading Buffer and then incubate the mixture at 98℃ for at least 10min.
11. Western blot.

The assay of human IL-10
    Since we were targeting the medical use of IL-10, it was very important to know the bio-activity and the expressing efficiency of IL-10 in our bacteria strain. We used human IL-10 ELISA kit (BOSTER Biological Technology co.Itd) to do the activity assay. It was notable that all our samples showed great IL-10 bio-activity, and the expressing level was super high (Fig. 15). After incubating at 16℃ for 20h, the concentration of IL-10 in the culture could be 23 ng/ml (Fig. 16).

Figure 16. A. Standard curve of human IL-10. B. CFUs result of activity assay using human IL-10 ELISA kit. CFUs were inoculated to LB medium followed by 12h incubation at 37℃. After induction of IPTG, the culture was incubated at 16℃ for 20h. Cells and medium were collected and ready for assay. The volume of culture for whole protein was 10ml while the volume of culture for secret protein was 50ml.
4. Kill switch
    This year, we made some improvements to our kill-switch system so as to palliate the symptom of severe MazF leakage problem. We introduced MazE to reduce the toxicity of MazF.
    At first, we measured the survival rates of the bacteria transformed with single MazF protein. We transformed the constructed PcspA-mazF plasmid constructed last year into E. coli BL21 and cultured at 37°C for 11 hours. We then cultured respectively overnight at 16 °C and 37°C. After diluting to $OD_{600}$=0.02 on the next day, the cells were cultured at 16 °C (Fig. 17A) or 37°C (Fig. 17B) and the OD60 value was measured every hour for 11 hours. Finally, we got the growth curves. By analyzing the growth curve, it showed that the survival rate of cells reaching 9.12% at 16°C and the survival rate of cells reaching 34.15% at 37°C.

Figure 17. The effect of our killer gene under different conditions. A, B. The effect of empty vector, PcspA-mazF on the growth of E. coli at different temperature (A,16°C; B,37°C) .
    In the next period, we constructed two new plasmids, one utilizing PT7 as promoter, pet28a as the vector and mazE as target gene, the other one utilizing PcspA as promoter pCold as the vector and mazF as target gene (Fig. 18). Then we co-transformed the two plasmids, PT7-mazE and PcspA-mazF, into E. coli BL21. At last, we measured the survival rate of cells transformed with mazEF system at 37°C and calculated the survival rate which reached 88.06% (Fig. 19). The result demonstrated that introducing MazE can increase the survival rates of bacteria cultured at 37 °C.
Figure 18. Kill switch module construction. A. The construction of PcspA-mazF plasmid. PcspA, the cold shock promoter. mazF, the toxin gene from E. coli. B. The construction of PT7-mazE plasmid. mazE, the anti-toxin gene from E. coli.
Figure 19. The effect of our killer gene under different conditions. A, B. The effect of empty vector, mazF and co-transformed mazE-mazF system on the growth of E.coli at different temperatures (A,16°C; B,37°C).
5. Intergrated experiments
PyeaR-hlL10
Figure 20. Diagram of PyeaR-hIL10 in pCDFDuet-1. Promoter yeaR is a nitric oxide inducible promoter. hIL-10 is one of our target gene.
    In order to see whether the nitric oxide sensor would work when it came to expressing a certain kind of protein, we added the IL-10’s coding gene downstream (Fig. 20) and demonstrated the immunoblotting to show the result (Fig. 21). It proved that the expression level could be induced when adding nitric oxide.
Figure 21. Protein expressing of human IL-10 gene which is transformed into E. coli BL21 strain and induced by nitric oxide. After induction of nitric oxide, the culture was incubated at 37°C for 2 hours. Immunoblotting demonstrated that the expression level of hIL-10. In order to equate the total number of cells in each lane, dilution was made to keep each sample at the same $OD_{600}$ and 10μL of each sample was uploaded to each lane.
Amplifier-hIL10
    To make sure that amplifier was able to strengthen the expression of gene downstream to promoter PhrpL (Fig. 22). A Coomassie brilliant blue was demonstrated that more protein at 35kDA, which should be hIL-10, was witnessed when introducing hIL-10 gene downstream of promoter PhrpL (Fig. 23).
Figure 23. Protein expressing of human hIL-10 gene which was transformed into E. coli BL21 strain. After induction of IPTG, the culture was incubated at 37℃ for 2 hours. A Coomassie brilliant blue showed the expression level of hIL-10. In order to equate the total number of cells in each lane, dilution was made to keep each sample at the same $OD_{600}$ and 15μL of each sample is uploaded to each lane.

    Secretory tag YebF was introduced to secret the protein, human IL-10 (Fig. 13). Immunoblotting was demonstrated that the secretion of humanIL-10. Expressing vector was constructed last year. Plasmid pCDFDuet-1 harboring YebF and human IL-10 gene was transformed into E. coli BL21 strain. After induction of IPTG, we span down the cells, getting them eliminated using filters. Secreted protein was collected from the medium by salting out with ammonium sulfate. The molecular weight of protein YebF-hIL-10 was 31.59 kDa (Fig. 14).
Figure 1. Diagram for yeaR based NO sensor system in pCDFDuet-1. PyeaR, a promoter which is sensitive to NO. Luciferase, reporter gene. Terminator B0010/B0012, double terminator.
Figure 10. Protein expression of myrosinase gene in E. coli BL21 strain. After induction of IPTG, the culture was incubated at 37℃ for overnight. The concentration of protein loaded on these two lanes were different.
Figure 14. Protein expression of human IL-10 gene in E. coli BL21 strain. After induction of IPTG at final concentration of 1mM, the culture was incubated at 37℃  for 12h. Bacterial cells were eliminated using centrifugation and then filters. The volume of cells loaded in the first four lanes was slightly different.
Figure 22. Diagram of amplifier-hIL10 in pCDFDuet-1. HrpR and HrpS amplifier system was used as a booster to boost the expression level of gene downstream promoter PhrpL. Protein HrpR and HrpS could bind together to induce the PhrpL which strengthens the expression of downstream gene YebF-hIL10.

u541_menu
NEU-CHINA
Figure 15. The result of activity assay using human IL-10 ELISA kit. Wells C1-C8 were standard human IL-10 protein sample with concentration at 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6pg/ml, 7.8pg/ml and 0pg/ml respectively. Wells B1-B4 were serial dilution of whole protein sample 1 with 1x, 10x, 100x and 1000x, while B5-B8 were serial dilution of whole protein sample 2. Wells A1-A3 were serial dilution of secret protein sample 1 with 1x, 10x and 100x, while wells A4-A6 were serial dilution of secret protein sample 2. Well A7 and A8 were control wells. Sample 1 and sample 2 were cultured from different CFUs after transforming expressing vector into BL21 strain.