Coding

Part:BBa_K3078004

Designed by: Yu Ma   Group: iGEM19_Jilin_China   (2019-10-14)
Revision as of 02:52, 21 October 2019 by Nashor (Talk | contribs)

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).

B13-1

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.

B13-2

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


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]


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Parameters
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