Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.
BACKGROUND
Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.


RESULTS
Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed beta-lactamase (DeltaW290) selection cassette contains a segment inside the beta-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests.


CONCLUSIONS
Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway or ligase-independent cloning (LIC) .
Olieric
Natacha
N
Paul Scherrer Institut, Biomolecular Research, Villigen PSI, Switzerland.
Kuchen
Melanie
M
Wagen
Sandro
S
Sauter
Marion
M
Crone
Stephanie
S
Edmondson
Sonia
S
Frey
Daniel
D
Ostermeier
Christian
C
Steinmetz
Michel O
MO
Jaussi
Rolf
R
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011994:Recombinant Proteins; D004247:DNA