Part:BBa_K2718022
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.
Purification
Our biobrick have his-tag in c-ter ,we can purify with a nickel column. After production, we break ours cells, and with lysate we purify on "Akta pure" on Histrap column (GE healthcare). We used like buffer 50 Mm Tris-HCl (pH=8), 50 mM NaCl. After fixation of chitinase on column, we used solution (buffer with 500 mM imidazolusing) 0 to 100% gradient to elute the column, we collect flowthrough gradually in 1 mL fractions. For more information, please visit our [http://2018.igem.org/Team:Aix-Marseille/Protocols wiki]
'image colonne elution'
We see one graph, one spike may correspond to the chitinase. Subsequently, to verify our purification we made SDS-PAGE
Fig 3. SDS-PAGE (12%) after purification on Akta pure with nickel colomn
We can see chitinase on fraction n°6 to fraction n°10 at aproximatively 30 kDa, that's correspond to chitinase6-his. Nonetheless, our chitinase is not pure and purification protocol can be improve,maybe by using an other nickel column, or by changing buffer, the his-tag appear efficient for purification.
Activity test
We used test, based on Schale's procedure to mesure reducing sugar produce by chitinase action on chitine. So we mesure N-acetyl-G-glucosamine thanks to this colorimetric reaction, when reduced sugar are producted by chitinase, the absorbance decrease. For more information about protocol see our [http://2018.igem.org/Team:Aix-Marseille/Results wiki].
Fig 4: Chitinase Activity test based on Schale's procedure
We encounter one major issue for test, it was difficult to dissolve chitin sample. It was problematic to pipet correctly, so there is problems with reproducibility . That semms explain we have results at 30 min and at 60 min it's not significant. Moreover, due to limited time, we didn't make other experiments to improve results. Nevertheless, we may conclude that our chitinase seems have activity
Design notes
This chitinase exists with:
- with only coding sequence BBa_K2718020
- with coding sequence and His-tag BBa_K2718021
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
- 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 230
- 1000COMPATIBLE WITH RFC[1000]
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