Difference between revisions of "Part:BBa K523000"

Revision as of 15:11, 6 September 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 part is mostly intended as a cloning vector; see below.

The part can also probably be used simply as a (detectable) promoter. If added upstream of an RBS and coding sequence, a polycistronic transcript will be generated with LacZ followed by the other gene.

Usage as a cloning vector

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 allows K523000 to be used as a cloning vector for creating new BioBricks in the following way: 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 the new part would circularise.

Procedure

To use as a vector for cloning a new part, first PCR the source gene so as to add a BglII site (agatct) upstream of the gene, and a t followed by a SpeI site (tactagt) downstream of it. Then follow this procedure...

Replate the colonies, then miniprep them and carry out verification sequencing. You should now have an RFC10-compatible BioBrick with a full prefix and suffix. The first four bases of the part will be atct. For an example of a BioBrick produced in this way, see BBa_K523002.

Sequence and Features