Difference between revisions of "Part:BBa K523000"

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This 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.
 
This 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.
  
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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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 (T ACTAGT) downstream of it. Then follow the following procedure...
<span class='h3bb'>Sequence and Features</span>
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[[Image:K523000 diagram.png]]
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This produces an RFC10-compatible BioBrick.
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===Sequence and Features===
 
<partinfo>BBa_K523000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K523000 SequenceAndFeatures</partinfo>
  

Revision as of 14:31, 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 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 the new part would circularise.

This 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 and Biology

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 (T ACTAGT) downstream of it. Then follow the following procedure...

K523000 diagram.png

This produces an RFC10-compatible BioBrick.

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]