Difference between revisions of "Part:BBa K523000"

Revision as of 22:04, 28 June 2011

Plac + lacZ; with BglII site

This is based on part BBa_J33207. Like that part, it contains the lac promoter and codes for 76 N-terminal residues of LacZ (Β-galactosidase), a peptide that complements the lacZ-delta-M15 mutation in common lab strains of E. coli. If grown on Xgal and IPTG, colonies with this part will be blue.

The first four bases of the sequence add a BglII site (BglII sites have 6 bases, but the first 2 are provided by the standard BioBrick prefix). This enables an easy method of using PCR to create new BioBricks (e.g. from natural sources): the forward primer for the new BioBrick should have a BglII site added, and the reverse primer should have a SpeI site added.

The PCR product and the plasmid containing this part can then both be digested with BglII and SpeI. After purification, mixing, ligation, and transformation, colonies with plasmids that have successfully gained the new BioBrick will be white, while those still containing the lacZ part will be blue (on Xgal+IPTG plates).

One might ask: why not use the standard flanking BioBrick sites XbaI and SpeI for this procedure? The answer is that these produce compatible sticky ends and so, upon digestion, the new part will circularise.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 The first four bases of the sequence add a BglII site (BglII sites have 6 bases, but the first 2 are provided by the standard BioBrick prefix). This enables an easy method of using PCR to create new BioBricks (e.g. from natural sources): the forward primer for the new BioBrick should have a BglII site added, and the reverse primer should have a SpeI site added.
-The PCR product and the plasmid containing this part can then both be digested with BglII and SpeI. After mixing, ligation, and transformation, colonies with plasmids that have successfully gained the new BioBrick will be white, while those still containing the lacZ part will be blue (on Xgal+IPTG plates).
+The PCR product and the plasmid containing this part can then both be digested with BglII and SpeI. After purification, mixing, ligation, and transformation, colonies with plasmids that have successfully gained the new BioBrick will be white, while those still containing the lacZ part will be blue (on Xgal+IPTG plates).
 One might ask: why not use the standard flanking BioBrick sites XbaI and SpeI for this procedure? The answer is that these produce compatible sticky ends and so, upon digestion, the new part will circularise.