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Merck KGaA
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15 febrero 2012
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pCDFDuet™-1 is designed for the coexpression of two target genes. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). The vector also carries the pCloDF13 replicon, lacI gene and streptomycin/spectinomycin resistance marker. This vector can be transformed into the same cell with pETDuet™-1, pACYCDuet™-1, and pRSFDuet™-1 or pCOLADuet™-1 for the coexpression of up to eight target genes. ORFs inserted into MCS-1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer. ORFs inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer.
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  <p>LaneM: Trail Mix™ Protein Markers; Lane1: pACYDuet-1:β-gal + Fluc; Lane2: pETDuet-1:GST/GUS + GFP; Lane3: pRSFDuet-1:Nus/hlFNγ + S•Tag/T4 PNK; Lane4: pCDFDuet-1:GUS + His•Tag/MBP; Lane5: pACYDuet-1, pETDuet-1 combination; Lane6: pACYDuet-1, pRSFDuet-1 combination; Lane7: pACYDuet-1, pCDFDuet-1 combination; Lane8: pETDuet-1, pRSFDuet-1 combination; Lane9: pRSFDuet-1, pCDFDuet-1 combination; Lane10: pETDuet-1, pCDFDuet-1 combination; Lane11: pACYDuet-1, pETDuet-1, pRSFDuet-1 combination; Lane12: pACYDuet-1, pETDuet-1, pCDFDuet-1 combination; Lane13: pACYDuet-1, pRSFDuet-1, pCDFDuet-1 combination; Lane14: pETDuet-1, pRSFDuet-1, pCDFDuet-1 combination; Lane15: Uninduced control; Lane16: All four Duet vectors: all eight proteins</p> <p>The indicated constructs were transformed individually or together into BL21(DE3). Cultures were grown in TB plus phosphates plus glucose at 37°C to and Abs<sub>600</sub> between 1.0 and 1.2. Target protein expression was induced by adding IPTG to a final concentration of 1 mM. Cultures were harvested by centrifugation 2.5 h after induction. Lysates were produced by sonication using equal volumes of 1% SDS and 2X SDS sample buffer. Equivalent amounts of protein (based on harvest ABS) were analyzed by SDS-PAGE (4-20% gradient gel) and stained with coomassie blue. To maximize band separation, proteins smaller than 25 kDa were allowed to migrate off the gel.</p> |
