Part:BBa_K3078004
β-1,3-glucanase
β-1,3-glucanase protein coding region. β-1,3-glucanase can degrade biofilm.
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
We characterised β-1,3-glucanase by cloning it into pVE vector. Moreover, an signal peptide were added.
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 following agarose gel electrophoresis (Figure 1).
Figure 1. Digestion and agarose gel electrophoresis of pVE-β-GA.
To assess the expression of our β-1,3-glucanase, Congo Red experiment was used.
Congo Red has a strong chromogenic reaction with β-1, 3-glucan, and 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. The results of activity detection of Congo Red hydrolytic ring (Figure 2) showed that compared to the control, there was a larger size of transparent hydrolytic circle caused by β-1, 3-glucanase expressed in E. coli with pVE-β-GA.
Figure 2. Expression of pVE-β-GA. 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.
3. Conclusion
To assess the expression of our β-1,3-glucanase, Congo Red experiment was used.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 274
Illegal NgoMIV site found at 483
Illegal NgoMIV site found at 622 - 1000COMPATIBLE WITH RFC[1000]
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