Difference between revisions of "Part:BBa K1088016"

Revision as of 01:14, 4 October 2013

HRT2 prenyltransferase from Hevea Brasilianis (ara promoter with araC: arabinose inducible)

This part encodes the rubberproducing prenyltransferase HRT2 from the rubber tree Hevea brasiliensis. It uses isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to produce cis-1,4 polyisoprene natural rubber.

The part is designed to express the enzyme when induced with arabinose under glucose scarce conditions. Arabinose binds the ara promoter regulator, AraC, and arabinose bound AraC then activates the transcription of the ara promoter.

The coding region of araC with a constitutively active promoter, a strong RBS and a efficient terminator was added to the part to see if the expression from the ara promoter would be elavated compared to a similar construct (BBa_K1088024) not bearing the araC device.

Using Northern blot technique it was proved that the construct in E. coli K-12 MG1655 doesn't express the prenyltransferase when grown without arabinose. Upon addition of arabinose to the media prenyltranferase mRNA was detected. The addition of the araC device did not prove to elevate the expression level. See BBa_K1088017 for more details.

A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter. B) Northern blot result reflecting diagram.

In conclusion we proved that we can induce the expression by addition of arabinose.

A 3xFLAG tag is behind the HRT2 prenyltransferase, but ins't part of the protein (HRT2 has it's natural stop-codon).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1102
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1042
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 8: / Line 8: @@
 The coding region of araC with a constitutively active promoter, a strong RBS and a efficient terminator was added to the part to see if the expression from the ara promoter would be elavated compared to a similar construct (BBa_K1088024) not bearing the araC device.
-Using Northern blot technique it was proved that the construct in ''E. coli'' K-12 MG1655 doesn't express the prenyltransferase when grown without arabinose. Upon addition of arabinose to the media prenyltranferase mRNA was detected. The addition of the araC device did not prove to elevate the expression level. See experince for more details.
+Using Northern blot technique it was proved that the construct in ''E. coli'' K-12 MG1655 doesn't express the prenyltransferase when grown without arabinose. Upon addition of arabinose to the media prenyltranferase mRNA was detected. The addition of the araC device did not prove to elevate the expression level. See BBa_K1088017 for more details.
+https://static.igem.org/mediawiki/2013/e/ee/SDU2013_Part_BBa_K1088017.png
+A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter.
+B) Northern blot result reflecting diagram.
+In conclusion we proved that we can induce the expression by addition of arabinose.
 A 3xFLAG tag is behind the HRT2 prenyltransferase, but ins't part of the protein (HRT2 has it's natural stop-codon).