Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes.
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 beta-galactosidase alpha-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 alpha-complementation and use insertion of the synthetic DNA segment to restore alpha-complementation. The alpha-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 alpha-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.
Cronan
J E
JE
Department of Microbiology, University of Illinois, Urbana 61801.
Narasimhan
M L
ML
Rawlings
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003360:Cosmids; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D005696:Galactosidases / Q000235:genetics*; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000235:genetics*
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D009841:Oligonucleotides; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase