Difference between revisions of "Part:BBa K3078002"
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<h1>'''1. Usage and Biology'''</h1>
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<h5>
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<P style="text-indent:2em;">
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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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</p>
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</h5>
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<h1>'''2. Characterization'''</h1>
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<h4>'''2.1 Validation of pVE-&#946;-1,3-glucanase construction'''</h4>
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<h5>
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<P style="text-indent:2em;">
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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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</p>
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</h5>
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[[File:B131.png|600px|center|B131]]
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<center>
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Figure 1. Digestion and electrophoresis of pVE-&#946;-GA.
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</center>
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<h4>'''2.2 Expression of pVE-&#946;-1,3-glucanase '''</h4>
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<h5>
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<P style="text-indent:2em;">
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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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</p>
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</h5>
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[[File:B132.png|center|B132]]
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<center style="text-align:left;">
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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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</center>
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<h4>'''2.3 Degradation effect of &#946;-1,3-glucanase on biofilm '''</h4>
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<h5>
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<P style="text-indent:2em;">
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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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</p>
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</h5>
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[[File:B133.png|center|B133]]
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<center style="text-align:left;">
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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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</center>
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<h1>'''3. Conclusion'''</h1>
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<h5>
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<P style="text-indent:2em;">
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</p>
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</h5>
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===Functional Parameters===
 
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Revision as of 16:07, 21 October 2019

Msp1

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



1. Usage and Biology

β-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 β-1,3-glucanase, form Cellulosimicrobium cellulans, can degrade β-1,3-glucan. Therefore, this year, we decided use β-1,3-glucanase to disrupt the Candida biofilm matrix and increase the effect of the antimicrobial drug.


2. Characterization

2.1 Validation of pVE-β-1,3-glucanase construction

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 1).

B131

Figure 1. Digestion and electrophoresis of pVE-β-GA.



2.2 Expression of pVE-β-1,3-glucanase

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 E. coli with pVE-β-GA, indicating the expression of β-1,3-glucanase (Figure 2).

B132

Figure 2. 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 E. coli 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.


2.3 Degradation effect of β-1,3-glucanase on biofilm

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 3 shows, β-1,3-glucanase produced by our engineered bacteria has the effect of degrading biofilm. The supernatant of E. coli 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 3).

B133

Figure 3. 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 and 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.



3. Conclusion










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 274
    Illegal NgoMIV site found at 483
    Illegal NgoMIV site found at 622
  • 1000
    COMPATIBLE WITH RFC[1000]