Difference between revisions of "Part:BBa K2718022"

Revision as of 14:53, 13 October 2018

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 2). 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.

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]

@@ Line 5: / Line 5: @@
 ===Usage and Biology===
-This is an improvement of part [http://https://parts.igem.org/Part:BBa_K1913000 BBa_K1913000], a chitinase from <i>Seratia marcescens</i>. 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 2).
+This is an improvement of part [https://parts.igem.org/Part:BBa_K1913000 BBa_K1913000], a chitinase from <i>Seratia marcescens</i>. 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 2).
 A number of groups have previously attempted to produce chitinases,
-[http://https://parts.igem.org/Part:BBa_K622006 BBa_K622006],
+[https://parts.igem.org/Part:BBa_K622006 BBa_K622006],
-[http://https://parts.igem.org/Part:BBa_K110499 BBa_K110499],
+[https://parts.igem.org/Part:BBa_K110499 BBa_K110499],
-[http://https://parts.igem.org/Part:BBa_K1201000 BBa_K1201000],
+[https://parts.igem.org/Part:BBa_K1201000 BBa_K1201000],
-[http://https://parts.igem.org/Part:BBa_K1298001 BBa_K1298001],
+[https://parts.igem.org/Part:BBa_K1298001 BBa_K1298001],
-[http://https://parts.igem.org/Part:BBa_K1913000 BBa_K1913000] and
+[https://parts.igem.org/Part:BBa_K1913000 BBa_K1913000] and
-[http://https://parts.igem.org/Part:BBa_K2224001 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
+[https://parts.igem.org/Part:BBa_K2224001 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
-[http://https://parts.igem.org/Part:BBa_K2224001 BBa_K2224001] of team
+[https://parts.igem.org/Part:BBa_K2224001 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 <i>E. coli</i> have been unsuccessful the attempts with fungal enzymes have been more successful.