Difference between revisions of "Part:BBa K523000"

Revision as of 13:22, 7 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.

Usage as a promoter

The part can 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.

K523000 being used to prepare BBa_K523002. White colonies should have the correct sequence.

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 downstream of it (so: t actagt).

The gene must not contain any BglII sites or SpeI sites. However, genes containing EcoRI, XbaI, or PstI sites can be successfully cloned this way; afterwards site-specific mutagenesis must be carried out to remove them, so as to produce an RFC10-compatible BioBrick.

Primers for an imaginary gene might look like this:

• Forward: aaa agatct atg tat tcg gct aat gat
• Reverse: aaa actagt a tta tta gct ttc gtg gca gat cca

(The "aaa" bases are just to give the restriction enzymes room to work.)

After PCR, purify the PCR product and then follow this procedure... (or something similar. This is Edinburgh 2011's exact procedure. For detailed protocols, see the [http://openwetware.org/wiki/French_Lab Open Wetware: French Lab] page.)

Replate some white 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.

Parts produced in this way will also be compatible with Edinburgh's 2011 BioSandwich protocol, though its use is entirely optional.

Sequence and Features