Difference between revisions of "Part:BBa K3078002"
 
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<h1>'''1. Usage and Biology'''</h1>
 
<h1>'''1. Usage and Biology'''</h1>
 
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&#946;-1,3-glucan is one of the primary components in C. albicans biofilm EPS, which is important for Candida biofilm formation and resistance to stresses. The enzyme &#946;-1,3-glucanase, form Cellulosimicrobium cellulans, can degrade &#946;-1,3-glucan. Therefore, this year, we decided use &#946;-1,3-glucanase to disrupt the Candida biofilm matrix and increase the effect of the antimicrobial drug.  
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<i>C. albicans</i> in hypha phase can invade human epithelial cells and cause infection. In order to eliminate the hyphae of <i>C. albicans</i>, we choose the protein Msp1 with the function of destabilizing hyphae.
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Msp1 is a protein secreted by rhamnose GG and has chitinase activity. There is a large amount of chitin in the hypha cell wall of <i>C. albicans</i>, so Msp1 can hydrolyze chitin to achieve the effect of destabilising hyphae.
 
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<h1>'''2. Characterization'''</h1>
 
<h1>'''2. Characterization'''</h1>
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<h4>'''2.1 Validation of pVE-&#946;-1,3-glucanase construction'''</h4>
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<h4>'''2.1 Validation of Msp1 construction'''</h4>
 
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To verify the construction of pVE-&#946;-1,3-glucanase (pVE-&#946;-GA) which we generated, the digestion by SalI/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 1).
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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 1).
 
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[[File:B131.png|600px|center|B131]]
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[[File:Msp11.png|600px|center|Msp11]]
 
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Figure 1. Digestion and electrophoresis of pVE-&#946;-GA.
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Figure 1. Digestion and electrophoresis of pVE-Msp1.
 
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<h4>'''2.2 Chitinase activity of Msp1 '''</h4>
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<h4>'''2.2 Expression of pVE-&#946;-1,3-glucanase '''</h4>
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To assess the &#946;-1,3-glucanase expression of our construct, Congo Red experiment was used. Congo Red has a strong red chromogenic reaction with &#946;-1,3-glucan. In contrast, when &#946;-1, 3-glucan is decomposed into reducing monosaccharides by &#946;-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 &#946;-1,3-glucanase expressed in E. coli with pVE-&#946;-GA, indicating the expression of &#946;-1,3-glucanase (Figure 2).
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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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[[File:B132.png|center|B132]]
 
 
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Figure 2. Expression of &#946;-1,3-glucanase. Add 10 mg/mL Congo Red solution to LB medium containing &#946;-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 E. coli with pVE-&#946;-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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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(R2=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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[[File:Msp12.png|600px|center|Msp12]]
  
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<h4>'''2.3 Destabilization of <i>C. albicans</i> hyphae by Msp1'''</h4>
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<h4>'''2.3 Degradation effect of &#946;-1,3-glucanase on biofilm '''</h4>
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Crystal violet (CV) reduction method, which is commonly used for quantitative analysis of biofilm, was used to evaluate the antibiofilm activity of &#946;-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 3 shows, &#946;-1,3-glucanase produced by our engineered bacteria has the effect of degrading biofilm. The supernatant of E. coli with pVE-&#946;-GA diluted by one time is estimated to reach the effect of 0.5 μg/mL~2 μg/mL samples of standard &#946;-1,3-glucanase. The result demonstrated that &#946;-1,3-glucanase had disruption effect on mature biofilm with concentration-dependent manner(Figure 3).
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It is reported that the proportion of chitin is three times higher than that of yeast cell wall in the hypha cell wall of <i>C. albicans</i>. Due to chitinase activity of Msp1, we inferred that Msp1 can cause the inhibition and degradation of hypha cell wall.  
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To detect the inhibition ability of Msp1, <i>C. albicans</i> 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 2).
 
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[[File:B133.png|center|B133]]
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[[File:Msp1-3.png|600px|center|Msp1-3]]
 
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Figure 3. Degradation effect of &#946;-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-&#946;-GA and standard &#946;-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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Figure 2. Inhibition ability of Msp1. <i>C. albicans</i> 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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To further test the degradation ability of Msp1, <i>C. albicans</i> was firstly only cultured by medium with serum to produce hyphae and then we added the concentrated bacteria supernatant into the cultured <i>C. albicans</i>. Compared to the control, the hyphae became shorter, indicating that Msp1 exhibited the capacity of destabilizing hyphae (Figure 3).
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[[File:Msp1-4.png|600px|center|Msp1-4]]
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Figure 3. Degradation ability of Msp1. <i>C. albicans</i> 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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Msp1 can combine with lactic acid and some proteins secreted by <i>Lactobacillus</i> and synergistically abolish hyphal morphogenesis. We reasonably predict that Msp1 will have better effect when Lactobacillus jensenii is used as the chassis for our project in the future.
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<h1>'''3. Conclusion'''</h1>
 
<h1>'''3. Conclusion'''</h1>
 
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After verification of experiments，the results showed that MSP1 had chitinase activity and the ability of destabilizing <i>C. albicans</i> hyphae.
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===Functional Parameters===
 
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Latest revision as of 02:22, 22 October 2019

Msp1

Msp1 protein coding region. Msp1 is a protein with chitinase activity.

1. Usage and Biology

C. albicans in hypha phase can invade human epithelial cells and cause infection. In order to eliminate the hyphae of C. albicans, we choose the protein Msp1 with the function of destabilizing hyphae.

Msp1 is a protein secreted by rhamnose GG and has chitinase activity. There is a large amount of chitin in the hypha cell wall of C. albicans, so Msp1 can hydrolyze chitin to achieve the effect of destabilising hyphae.

2. Characterization

2.1 Validation of Msp1 construction

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 1).

Msp11

Figure 1. Digestion and electrophoresis of pVE-Msp1.

2.2 Chitinase activity of Msp1

Msp1 has the chitinase activity which can destabilize hyphae. Chitinase activity kit was used to detect the chitinase activity of Msp1.

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(R2=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.

Msp12

2.3 Destabilization of C. albicans hyphae by Msp1

It is reported that the proportion of chitin is three times higher than that of yeast cell wall in the hypha cell wall of C. albicans. 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, C. albicans 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 2).

Msp1-3

Figure 2. Inhibition ability of Msp1. C. albicans 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.

To further test the degradation ability of Msp1, C. albicans was firstly only cultured by medium with serum to produce hyphae and then we added the concentrated bacteria supernatant into the cultured C. albicans. Compared to the control, the hyphae became shorter, indicating that Msp1 exhibited the capacity of destabilizing hyphae (Figure 3).

Msp1-4

Figure 3. Degradation ability of Msp1. C. albicans 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.

Msp1 can combine with lactic acid and some proteins secreted by Lactobacillus and synergistically abolish hyphal morphogenesis. We reasonably predict that Msp1 will have better effect when Lactobacillus jensenii is used as the chassis for our project in the future.

3. Conclusion

After verification of experiments，the results showed that MSP1 had chitinase activity and the ability of destabilizing C. albicans hyphae.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1011
  • 1000
    COMPATIBLE WITH RFC[1000]