Team:TU Darmstadt/Notebook/sec1/K1602007

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K1602007 - Cis-Aconitate Decarboxylase with strong RBS (B0034-cadA)

Figure 1 PCR of RBS-cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).

The cadA-gene was synthesized by GeneArt and amplified through PCR using the cadA-RBS (FW) and cadA (REV) oligonucleotides to amplify it including the B0034 ribosomal binding site. The PCR product was verified by agarose gel electrophoresis and subsequently purified. The cadA amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. The ligation product was transformed into E.coli Top 10 by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by Sanger sequencing.