Difference between revisions of "Part:BBa I732400"

Revision as of 17:47, 25 October 2007

Promoter Family Member (U097NUL+D062NUL)

P_template1 serves as the truss for PCR cloning. Firstly, we extend both sides of the conservative region for transcriptional initiation of PlacUV5, including -35 box，-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:

They will never include the restriction enzyme cutting sites that will be involved in the whole study;
They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;
They will never present in complicated structures.

Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.

Then the promoter fragments are digested with XbaI and BamHI and cloned into repression-reporter plasmid, which contains lacZ alpha fragment and gfp under the promoter insertion site.

[[Image:USTC_ Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 158
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 18

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 __NOTOC__
 <partinfo>BBa_I732400 short</partinfo>
-P_template1 serves as the truss for PCR cloning. By respectively applying 7 different primers upstream and downstream, that is, 49 pairs of primers in total, we have got 49 different promoters. Then we made them digsted and loaded into a vector that has already possessed a lacZ-alpha fragment. In this way, we can read from the color of the clones on the plate whether the inserted promoter works or not.
+P_template1 serves as the truss for PCR cloning. Firstly, we extend both sides of the conservative region for transcriptional initiation of PlacUV5, including -35 box，-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as [https://parts.igem.org/wiki/index.php?title=Part:BBa_I732400 P_template1] as it is the template for the promoter family. These two non-sense sequence have three main characters:
+# They will never include the restriction enzyme cutting sites that will be involved in the whole study;
+# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;
+# They will never present in complicated structures.
+Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.
+Then the promoter fragments are digested with XbaI and BamHI and cloned into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.
-By this means, We can try seven different positions for Operator A and seven for Operator B, thus constructing 7*7 kinds of promoters which have actually constituted a specific promoter family. After transformed into the four test plasmids (pCR00, pCR01, pCR10, pCR11) together with a downstream double-reporter system, we will get to know the solo-repression and co-repression effects of the two repressors on the specific promoter.
+[[Image:USTC_PCRBuilding.png|thumb|400px]]
+[[Image:USTC_
 <!-- Add more about the biology of this part here
 ===Usage and Biology===