Template:Jilin China/Res C.js
/**
* * @authors lhx * @date 2019-05-30 16:25:38 * @version 1.1 */
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prospect:{
title:"Result"
},
part: [{
title: "Therapeutic System",
para: [
{
type: "word",
cont: "Bcam0581—Phase transformation inhibition",
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{
type: "word",
cont: "Validation of pVE-Bcam0581 construction",
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cont: "To verify the construction of pVE-Bcam0581 which we generated, the digestion by ApaI/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 1).",
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cont: "Expression of Bcam0581",
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cont: "To detect the expression of Bcam0581 in (ita)E. coli(itaed), the constructs were transformed into (ita)E. coli(itaed) BL21(DE3). Compared to the negative control, a distinct band at 37 kD was showed in pVE-Bcam0581(Figure 2).",
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cont: "Figure 2. Expression of Bcam0581. The bacteria were collected and ultrasonicated. The lysate was centrifuged and supernate was electrophoresed on the SDS-PAGE gel, followed by Coomassie brilliant blue staining.",
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{
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cont: "Characterization of BDSF by HPLC.",
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cont: "Bcam0581 can produce the diffusible chemical BDSF that inhibits the phase transformation of (ita)C. albicans(itaed). The standard of BDSF concentration at 212 nm was measured with HPLC and the standard curve of BDSF concentration was established (Figure 3A&3B). To detect the concentration of BDSF which is the catalyzed production of Bcam0581, the concentrated bacteria supernatant of mock control or Bcam0581 were measured. Compared with the control , the Bcam0581 supernatant had an obvious absorption peak with a peak area of 127352 mAU·s at the retention time of 12.7 min. After calculation with the standard curve in Figure 3B, the BDSF concentration was 7.39 μM in the concentrated Bcam0581 bacteria (Figure 3C). ",
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cont: "Figure 3. Characterization of BDSF by HPLC. A. HPLC analysis of BDSF standard with concentration of 1.25, 2.5, 5 or 10 mM. The maximum absorption peak was at 12.7 min. B. The standard curve of BDSF standard. The relationship between the peak area of BDSF standard HPLC results and BDSF in different concentrations: Peak area (mAU·s)=18443x(mM)-8870.2(mAU·s), R(sup)2(suped)=0.9953. C. HPLC of concentrated mock control or pVE-Bcam0581. The constructs were transformed into (ita)E. coli(itaed) BL21. The bacteria were collected and ultrasonicated. The supernate from lysate was 1000 fold concentrated.",
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{
type: "word",
cont: "Phase transformation inhibition effect of BDSF from Bcam0581",
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cont: "To verify the phase transformation inhibition effect of BDSF, the standards or Bcam0581 bacteria supernatant were co-cultured with (ita)C. albicans(itaed). As shown in Figure 4. C3, 7.5 μM BDSF standards could completely inhibit the growth of hyphae. In contrast to control, the effect of the Bcam0581 bacteria supernatant inhibition activity on phase transformation is evident.(Figure 4).",
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cont: "Figure 4. BDSF inhibition of the phase transformation of (ita)C. albicans(itaed). (ita)C. albicans(itaed) and the bacteria supernatant of control or Bcam0581 or BDSF standard were co-cultured in YPD medium supplementary with 10% serum in 96-well plate at 37 ℃ for 4 hours. After incubation, the hypha morphology was observed with inverted microscope. A, mock control; B, bacteria supernatant of Bcam0581; C1~C3, different concentration of 0.75, 3 or 7.5 μM standard.",
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{
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cont: "Msp1—Destabilization of hyphae",
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{
type: "word",
cont: "Validation of Msp1 construction",
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{
type: "word",
cont: "To verify the construction of pVE-Msp1 which we generated, the digestion by SalI/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 5).",
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cont: "Chitinase activity of Msp1",
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cont: "Msp1 has the chitinase activity which can destabilize hyphae. Chitinase activity kit was used to detect the chitinase activity of Msp1.",
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cont: "Table 1. Chitinase activity of Msp1. The chitinase activity was performed by the standard protocol. In brief, after incubation, the absorbance was measured at 540 nm. The concentrated bacteria supernatant pVE5523 was used as control. Then the chitinase activity was calculated according to the standard curve, y=0.3331x-0.2557(R(sup)2(suped)=0.991). Production of 1 μmol N-acetylglucosamine in 1 mL culture medium decomposes chitin in 1 hour at 37 ℃ is defined as an active unit.",
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{
type: "word",
cont: "Destabilization of (ita)C. albicans(itaed) hyphae by Msp1",
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cont: "It is reported that the proportion of chitin is three times higher than that of yeast cell wall in the hypha cell wall of (ita)C. albicans(itaed). Due to chitinase activity of Msp1, we inferred that Msp1 can cause the inhibition and degradation of hypha cell wall. To detect the inhibition ability of Msp1, (ita)C. albicans(itaed) was co-cultured with Msp1 to observe the change of hyphae. Compared to the control, the hyphae were inhibited, which indicated that Msp1 exhibited the capacity of destabilizing hyphae (Figure 6).",
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cont: "Figure 6. Inhibition ability of Msp1. (ita)C. albicans(itaed) and the supernatant of control or Msp1 were co-cultured in YPD medium supplementary with 10% serum in 96-well plate at 37 ℃ for 4 hours. After incubation, the plate was observed with inverted microscope. A, mock control; B, Msp1.",
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cont: "To further test the degradation ability of Msp1, (ita)C. albicans(itaed) was firstly only cultured by medium with serum to produce hyphae and then we added the concentrated bacteria supernatant into the cultured (ita)C. albicans(itaed). Compared to the control, the hyphae became shorter, indicating that Msp1 exhibited the capacity of destabilizing hyphae (Figure 7). ",
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cont: "Figure 7. Degradation ability of Msp1. (ita)C. albicans(itaed) was cultured in YPD medium supplementary with 10% serum in 96-well plate at 37 ℃. 4 hours later, the supernatant of control or Msp1 was added into the plate respectively. After another 3-hour incubation, the plate was observed with inverted microscope. A, mock control; B, Msp1. ",
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cont: "Msp1 can combine with lactic acid and some proteins secreted by (ita)Lactobacillus(itaed) and synergistically abolish hyphal morphogenesis. We reasonably predict that Msp1 will have better effect when (ita)Lactobacillus jensenii(itaed) is used as the chassis for our project in the future. ",
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{
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cont: "β-1,3-glucanase--Biofilm Degradation",
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{
type: "word",
cont: "Validation of β-1,3-glucanase construction",
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{
type: "word",
cont: "To verify the construction of pVE-β-1,3-glucanase (pVE-β-GA) which we generated, the digestion by SalI/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 8).",
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cont: "Expression of β-1,3-glucanase",
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cont: "To assess the β-1,3-glucanase expression of our construct, Congo Red experiment was used. Congo Red has a strong red chromogenic reaction with β-1,3-glucan. In contrast, when β-1, 3-glucan is decomposed into reducing monosaccharides by β-1,3-glucanase, the hydrolyzed region forms a pale yellow transparent hydrolytic circle. Compared with control, there was a larger size of transparent hydrolytic circle caused by β-1,3-glucanase expressed in (ita)E. coli(itaed) with pVE-β-GA, indicating the expression of β-1,3-glucanase (Figure 9).",
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cont: "Figure 9. Expression of β-1,3-glucanase. Add 10 mg/mL Congo Red solution to LB medium containing β-1,3-glucan substrate (0.1 g/100 mL) at a ratio of 1:100. 100 μL supernatant obtained by centrifugation after ultrasonic crushing of (ita)E. coli(itaed) with pVE-β-GA is added to the Oxford cup, and the pVE empty vector bacteria supernatant is used as the control. Stand at 37 ℃ for 24 hours.",
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{
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cont: "Degradation effect of β-1,3-glucanase on biofilm",
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cont: "Crystal violet (CV) reduction method, which is commonly used for quantitative analysis of biofilm, was used to evaluate the antibiofilm activity of β-1,3-glucanase. Under the condition of using bacteria with pVE5523 vector(pVE vector) to exclude the influence of bacterial substances on the staining results, as Figure 10 shows, β-1,3-glucanase produced by our engineered bacteria has the effect of degrading biofilm. The supernatant of (ita)E. coli(itaed) with pVE-β-GA diluted by one time is estimated to reach the effect of 0.5 μg/mL~2 μg/mL samples of standard β-1,3-glucanase. The result demonstrated that β-1,3-glucanase had disruption effect on mature biofilm with concentration-dependent manner(Figure 10).",
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cont: "Figure 10. Degradation effect of β-1,3-glucanase on biofilm. Biofilm formed in RPMI 1640 medium for 48 hrs. Mature biofilm was treated with RPMI 1640 medium, bacteria supernatant of pVE vector or pVE-β-GA or standard β-1,3-glucanase in different concentrations (0.5, 1 and 2 μg/mL) for another 24 hrs. Values obtained are given as the percentage of biofilm. The experiment was performed three times in triplicate. *, P < 0.05 from mock control using Student’s t test. △, P < 0.05.",
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{
type: "word",
cont: "LL-37--Killing (ita)C. albicans(itaed)",
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{
type: "word",
cont: "Validation of LL-37 construction",
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cont: "To verify the construction of pVE-LL-37 which we generated, the digestion by BglII/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 11).",
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cont: "Expression of pVE-LL-37",
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cont: "To detect the expression of pVE-LL-37 in (ita)E. coli(itaed), the constructs were transformed into BL21. Compared to the mock, there was a small size band shown in pVE-LL-37 (Figure 12). ",
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cont: "Figure 12. Expression of pVE-LL-37. The bacteria were collected and ultrasonicated. The lysate was centrifuged and supernate was electrophoresed on the Tricine-SDS-PAGE gel, followed by Fast silver staining. The LL-37 peptide was synthesized as positive control. ",
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{
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cont: " Candidacidal effect of LL-37",
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{
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cont: "To assess the candidacidal activity of LL-37, the spot assay was performed. As shown in Figure 13, (ita)C. albicans(itaed) viability after LL-37 treatment was visually compared with that of control cells (no LL-37 treatment). We found that cell viability was more sensitive to LL-37.",
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cont: "Figure 13. Candidacidal activity of LL-37. (ita)C. albicans(itaed) were collected and suspensions were resuspended to 10(sup)5(suped) cells/mL. 10 µL (ita)C. albicans(itaed) suspension and 20 µL sterile BL21-transformed bacteria were gently mixed. After a 10-min incubation, the mixture was spotted onto the SDA agar plates. Cell viability was detected after incubation at 30 ℃ for 18 hrs.",
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cont: "To further validate the candidacidal effect of LL-37, (ita)C. albicans(itaed) suspensions were incubated with it. After incubation, LL-37 caused an immediate increase in PI fluorescence, indicating that LL-37 can kill (ita)C. albicans(itaed) (Figure 14)",
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] }, { type: "word", cont: "Figure 14. Candidacidal activity of LL-37. 1.5 µM PI (propidiumiodide) was added into (ita)C. albicans(itaed) suspensions (10(sup)6(suped) cells/mL, 1 mL) and serially BL21-transformed bacteria supernatant (2 mL) mixture. The whole system was incubated at 30 ℃. During the 1 h incubation, the fluorescence intensity was measured every 5 min at the excitation wavelength λexc 544 nm and emission wavelength λem 620 nm. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test.", class: "np6" }, { type: "word", cont: "Conclusion", class: "np3" }, { type: "word", cont: "In conclusion, we used BDSF, Msp1, β-1,3-glucanase and LL-37 in our therapeutic system to kill (ita)C.albicans(itaed). According to the results of our verification test, all of them are useful when working individually. It convinced that our engineered bacteria could work successfully and achieve our purpose in curing VVC by efficient biotherapy.", class: "np5" },
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}, { title: "Suicide System", para: [
{
type: "word",
cont: "TetR-T4 holin",
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{
type: "word",
cont: "Validation of pVE-TetR-T4 holin construction",
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cont: "To verify the construction of pVE-TetR-T4 holin which we generated, the digestion by SmalI was performed by a standard protocol followed by agarose gel electrophoresis (Figure 15).",
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cont: "Function of pVE-TetR-T4 holin",
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cont: "T4 holin is expressed after the suicide system is activated, which will lead to the non-specific damage of bacterial cytoplasmic membrane, forming stable transmembrane pores, and thus causing the death of bacteria.",
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type: "word",
cont: "When there is no tetracycline, P(sub)tet(subed) promoter will be inhibited by TetR, T4 holin will not be expressed and the bacteria survive. On the contrary, when tetracycline is added, TetR disinhibits P(sub)tet(subed) and T4 holin is expressed, forming a stable transmeminal pore, leading to bacterial death. As the results indicated, after 24 hrs of 50 nM atc treatment, over 50% bacteria were lysed and dead, which attested that suicide system could work successfully (Figure 16).",
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] }, { type: "word", cont: "Figure 16. Verification of the suicide system. The plasmid was transformed into (ita)E. coli(itaed) BL21, cultured overnight, and diluted to OD(sub)600(subed) 0.04. Anhydrotetracycline (atc) was added at OD(sub)600(subed) 0.9 to make the final concentration of atc reach 27 nM to measure at the indicated time. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test. △, P < 0.05.", class: "np6" }, { type: "word", cont: "Conclusion", class: "np3" }, { type: "word", cont: "As adding aTc to the culture medium with the engineered bacteria, we observed that most of them were killed and we detected T4 holin in the supernatant of the culture medium, which meant that our design of suicide system was successful.", class: "np5" }, ]
}, { title: "Sensing system", para: [
{
type: "word",
cont: "HpaX-HpaA-sfGFP",
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{
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cont: "Validation of HpaX-HpaA-sfGFP construction",
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{
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cont: "To verify the construction of pVE-HpaX-HpaA-sfGFP which we generated, the digestion by AgeI or XmaI was performed by a standard protocol followed by electrophoresis (Figure 17).",
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cont: "Figure 17. Digestion and electrophoresis of pVE-HpaX-HpaA-sfGFP. pVE-HpaX-HpaA-sfGFP can be digested into 1446 bp band and 900 bp band by AgeI and XmaI respectively. And it was verified by AgeI electrophoresis, showing that our construction was success.",
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{
type: "word",
cont: "Validation of pVE-HpaX-GFP construction",
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{
type: "word",
cont: "To verify the construction of pVE-HpaX-sfGFP which we generated, the digestion by AgeI or XmaI was performed by a standard protocol followed by electrophoresis (Figure 18).",
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{
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] }, { type: "word", cont: "Figure 18. Digestion and electrophoresis of pVE-HpaX-sfGFP. pVE-HpaX-sfGFP can be digested into 1446 bp and 11369 bp band by AgeI and XmaI respectively. And it was verified by AgeI electrophoresis, showing that our construction was success.", class: "np6" },
{
type: "word", cont: "Expression of pVE-HpaX-HpaA-sfGFP", class: "np3" },
{
type: "word", cont: "To detect the expression of pVE-HpaX-HpaA-sfGFP in (ita)E. coli(itaed), the constructions were transformed into (ita)E. coli(itaed) BL21(DE3). When the OD(sub)600(subed) of the culture reached 0.3, 4-HPA of gradient was added. However, according to our results, compared with the group without 4-HPA, the experimental group with high concentration of 4-HPA did not show significant fluorescence (Figure 19).", class: "np5" },
{
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] }, { type: "word", cont: "Figure 19. Fluorescence intensity of sfGFP under different concentration of 4-HPA. The emission intensity at 528 nm were measured at the excitation wavelength of 485 nm. Then measure OD(sub)600(subed). After that, measure these values at the 18(sup)th(suped) hour.", class: "np6" }, { type: "word", cont: "Conclusion", class: "np3" }, { type: "word", cont: "We used the HpaA-HpaX system in our project as the sensing system. As designed, we cultured the bacteria in the liquid LB medium with different concentration of 4-HPA solution. Although the unfortunate results are unpleasing, we have the confidence to get the positive data in the nearly future, with the reference and model. ", class: "np5" },
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}, { title: "Summary", para: [
{ type: "word", cont: "We successfully constructed and characterized our parts. Sufficient experiment results proved combination therapy was certainly more advantageous than single therapeutic factors' effect, pithily and convincingly. The suicide system guaranteed the safety of engineered bacteria and sensing system may react with (ita)C. albicans(itaed) density intelligently.", class: "np5" }
]
},
]
}