Insertional restoration of β-galactosidase α-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes

John E. Cronan, Meena L. Narasimhan, Merriann Rawlings

Research output: Contribution to journalArticlepeer-review


The assembly of synthetic genes from oligodeoxynucleotides can be an inefficient process. Upon ligation of a synthetic assembly into a plasmid vector and transformation of an Escherichia coli host, it is often found that only a minor fraction of the putative recombinant plasmids contains synthetic sequences. Moreover, the synthetic sequences cloned are often altered versions of those originally designed. We have designed a biological test to detect those plasmids that contain synthetic sequences of the proper length, termini and reading frame. The test is the reversal of the β-galactosidase α-complementation (blue-to-white) test used to detect the insertion of DNA segments into the polylinker sequences of the phage M13 mp, plasmids pUC, and related vectors. We begin with a modified vector defective in α-complementation and use insertion of the synthetic DNA segment to restore α-complementation. The α-complementation activity of the original vector (e.g., pUC18) was first abolished by a frameshift or DNA insertion within the polylinker sequence of the lacZ' gene segment. The α-complementation was then restored by insertion of the synthetic DNA sequence between the cohesive ends generated by digestion of two polylinker restriction sites. Formation of blue colonies requires the insertion of a DNA segment of appropriate length and termini to reconstruct the lacZ' open reading frame and thus is much more selective than the usual insertional inactivation strategy. We show that this 'insertional restoration' screening method markedly enhances the proper assembly of synthetic genes and describe manipulations to readily and reliably frameshift various polylinker sequences.

Original languageEnglish (US)
Pages (from-to)161-170
Number of pages10
Issue number1
StatePublished - Oct 15 1988


  • M13mp phage vectors
  • Recombinant DNA
  • frameshift
  • oligodeoxyribonucleotides
  • pTZ phagemid vectors
  • pUC plasmid vectors
  • polylinkers
  • transposons

ASJC Scopus subject areas

  • Genetics


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