Difference between revisions of "Part:BBa K1913000"

Revision as of 10:09, 14 October 2016

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 al^{<a href="#referenceschi">1</a>} 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 structure^{<a href="#referenceschi">2</a>}.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Characterization of chitinases

ChiA and ChiB were cloned into Escherichia coli BL21 and expressed using BioBrick devices <a href="https://parts.igem.org/Part:BBa_K1913002">BBa_K1913002</a> and <a href="https://parts.igem.org/Part:BBa_K1913003">BBa_K1913003</a> respectively. SDS-PAGE was performed to analyse the

[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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 <p>Chitinases from <i>Serratia marcescens</i> are very well characterized, as <i>Serratia marcescens</i> is considered a model organism for chitin degradation. This <i>chiA</i> BioBrick is based on the chitinases produced by <i>Serratia marcescens</i> strain GEI, a strain which causes high mortality in the parasitic honeybee mite <i>Varroa destructor</i>. Tu et al<sup><a href="#referenceschi">1</a></sup> cloned the chitinases into <i>Escherichia coli</i>, purified them and showed that ChiA and ChiB acted synergistically to increase mite mortality. </p>
 <h2>Structure and function</h2>
-<p>Both <i>ChiA</i> and <i>ChiB</i> 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. <i>ChiA</i> and <i>ChiB</i> 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 structure<sup><a href="#referenceschi">2</a></sup>. </p>
+<p><i>ChiA</i> and <i>ChiB</i> 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. <i>ChiA</i> and <i>ChiB</i> 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 structure<sup><a href="#referenceschi">2</a></sup>. </p>
 <span class='h3bb'>Sequence and Features</span>