Part:BBa_K1913000
chiA for Varroa destructor
Serratia marcescens GEI chiA protein coding region optimized for BioBrick use. This part is useful for breaking down chitin polymers. The strain of origin has been isolated from the Chinese honey bee and showed virulence towards Varroa destructor. Chitinases A and B from the strain were found to have miticidal activity. ChiA has both exochitinase and endochitinase activity. This protein is known to show synergistic activity with ChiB. ChiA can potentially be used to control Varroa destructor mite populations, as mite mortality increases when the mites are subjected to filter paper incubated with purified chiA.
Usage and Biology
Introduction
Chitinases from Serratia marcescens are very well characterized, as Serratia marcescens is considered a model organism for chitin degradation. This chiA BioBrick is based on the chitinases produced by Serratia marcescens strain GEI, a strain which causes high mortality in the parasitic honeybee mite Varroa destructor. Tu et al1 cloned the chitinases into Escherichia coli, purified them and showed that ChiA and ChiB acted synergistically to increase mite mortality.
Structure and function
ChiA and ChiB are modular enzymes with both a carbohydrate binding domain and a distinct catalytic domain. They can display both endochitinase and exochitinase activity. Endochitinase activity means that the enzymes cleave randomly inside the long chitin polymers, while exochitinase activity only targets the polymer's ends. ChiA and ChiB are both processive enzymes with exochitinase activity, but they can also show endochitinase activity if the substrate is accessible enough. Their carbohydrate binding domains are oriented in opposite directions and they each attack the chitin polymers from opposite ends, which seems to explain their synergistic action. However, most studies on chitinase function have been done on synthetic substrates rather than natural substrates such as shrimp chitin. It is therefore not clear how the specificity of strain GEI's chitinases is related to substrate structure2.
Improvment for purification and activity - Aix-Marseille 2018
Group : iGEM18_Aix-Marseille
We made a chitinase comes from fungus, optimize for production in E.coli, with an His-tag for purification and with an activity. For more informations see BBa_K2718022
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization of chitinases
ChiA and ChiB were cloned into Escherichia coli BL21 and expressed using BioBrick devices BBa_K1913002 and BBa_K1913003 respectively. SDS-PAGE was performed to analyse the constructs for expression of the chitinases, but no significant expression could be observed (Figure 1). Expression was also tested with colloidal chitin plates, but this assay failed to yield results.
Figure 1. Photo of a 10% BioRad MiniProtean gel loaded with protein extracts from BL21 constructs with chitinase devices. Device BBa_K1913002 is supposed to express a 81.5 kDa protein, while BBa_K1913003 should express a 76 kDa protein and BBa_K1913004 should express both. They were induced with 0%, 0.2% or 1% arabinose. The gel was run for 38 minutes at 50 mA together with a gel for the cellular debris (not shown).
[1] Tu, S., Qiu, X., Cao, L., Han, R., Zhang, Y., & Liu, X. (2010). Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite. Journal of invertebrate pathology, 104(2), 75-82.
[2] Vaaje‐Kolstad, G., Horn, S. J., Sørlie, M., & Eijsink, V. G. (2013). The chitinolytic machinery of Serratia marcescens–a model system for enzymatic degradation of recalcitrant polysaccharides. FEBS Journal, 280(13), 3028-3049.
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