Composite

Part:BBa_K2718022

Designed by: Dimitri Popoff Emeline Lemarie   Group: iGEM18_Aix-Marseille   (2018-08-27)
Revision as of 23:52, 17 October 2018 by Sowan (Talk | contribs) (→‎Purification)


IPTG inducible promoter with RBS Endochitinase 6-His

Usage and Biology

This is an improvement of part BBa_K1913000, a chitinase from Seratia marcescens. The part represents an improvement of this previous part as we are able to show production of the protein, purify the protein (figure 1), and preliminary experiments show some activity(figure 4). A number of groups have previously attempted to produce chitinases, BBa_K622006, BBa_K110499, BBa_K1201000, BBa_K1298001, BBa_K1913000 and BBa_K2224001, but to date, success has been very limited. Prior to our experiments, no group has been able to show production of significant amounts of the protein, no enzyme has been engineered to aid purification, and only one previous report has shown activity BBa_K2224001 of team SMS_Shenzhen in 2017 (they did not show significant amounts of protein and their protein was not engineered for purification). It is interesting to note that while attempts to produce bacterial and plant chitinases in E. coli have been unsuccessful the attempts with fungal enzymes have been more successful.

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To verify the functionality of this part we measured chitinase production after induction of E. coli (DH5a) at OD 0.8 with 1mm IPTG for 3 hours at 37°C.

Fig 1. (left) SDS-PAGE of chitinase production and (right) western blot revealed with anti-his antibody in NI (nor induced) and I (induced) E.coli cells.
This clearly shows that we are able to produce significant amounts of chitinase.

Purification

As our biobrick has a C-terminal his-tag, we are able to purify it with Histrap column (GE healthcare) using an "Akta pure" system. For detailed protocol, please visit our [http://2018.igem.org/Team:Aix-Marseille/Protocols#Protein_purification wiki]

T--Aix-Marseille--ChitinaseResults11.jpg

We see on graph, only one major peak was observed during elution which corresponds to the chitinase. To verify our purification we made SDS-PAGE analysis, .

--Aix-Marseille--ChitinaseResults_3_registry.jpg

Fig 3. SDS-PAGE analysis of chitinase after purification on Akta pure with Histrap column

See protocol in our [2018.igem.org/Team:Aix-Marseille/Protocols#Western_blot wiki]. We can see chitinase on fraction n°6 to fraction n°10 at aproximatively 30 kDa, this corresponds to the expected size of our protein. Nonetheless, our chitinase is not pure and purification protocol can be improved. In conclusion,the his-tag appears efficient for chitinase purification.

Activity test

In order to test the activity of purified chitinsas,we used [http://2018.igem.org/Team:Aix-Marseille/Protocols#Schales'_test] Schale's procedure. In this assay, chitinase is incubated with its substrate, chitin, which results in the production of N-acetyl-G-glucosamine. We measured abosobance at 420nm of the Schale's reagent. For more information about protocol see our[http://2018.igem.org/Team:Aix-Marseille/Protocols wiki].

T--Aix-Marseille--ChitinaseResults4.jpg

Fig 4: Chitinase Activity test based on Schale's procedure

We encounter one major issue for this test, as it was difficult to dissolve chitin sample. It was problematic to pipet correctly, so there are problems with reproducibility . That may explain why we see small but significant chitinase activity at 30 min, while the 60 min timepoint was not significant. Moreover, due to limited time, we didn't make other experiments to improve results. Nevertheless, we conclude that our purified chitinase seems to be active. For more informations about test, visit our [http://2018.igem.org/Team:Aix-Marseille/Experiments#Chitinase_activity_test wiki]

Design notes

This chitinase exists with:

This biobrick is in RFC 10 and RFC 25 standards with :

  • the prefixe in RFC 10 : GAATTC GCGGCCGC T TCTAGA G
  • and the suffixe in RFC 25 (contain Stop in frame): ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG 3'

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 230
  • 1000
    COMPATIBLE WITH RFC[1000]

Weiguo Fang, Bo Leng, Yuehua Xiao, Kai Jin, Jincheng Ma, Yanhua Fan, Jing Feng, Xingyong Yang, Yongjun Zhang, Yan Pei (2005).Cloning of Beauveria bassiana Chitinase Gene Bbchit1 and Its Application To Improve Fungal Strain Virulence Appl. Environ. Microbiol. Jan 2005, 71 (1) 363-370; DOI: 10.1128/AEM.71.1.363-370.2005

Rajasekhar, Pinnamaneni & Kalidas, P & Sambasiva Rao, K.R.S.. (2010). Cloning and Expression of Bbchit1 gene of Beauveria bassiana~!2009-03-20~!2010-04-10~!2010-06-30~!. The Open Entomology Journal. 4. 30-35. 10.2174/1874407901004010030.

Pedrini N, Ortiz-Urquiza A, Huarte-Bonnet C, Zhang S and Keyhani NO (2013) Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction. Front. Microbio. 4:24. doi: 10.3389/fmicb.2013.00024

Ferrari et al., A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection.Biotechnology for Biofuels 2014, 7:37

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