Part:BBa_K4075013

T7-LacO Promoter + RiboJ + RBS_0034 + mCherry + double Terminator

This part is made of a T7-LacO Promoter, Ribo J, RBS_0034, OmpA signal peptide, 6xHis-tag, followed by the sequence coding for the reporter gene mCherry, and ends in a double terminator. This device enables the expression of the fluorescent protein mCherry in Escherichia coli strains with T7 polymerase.

Assembly

The synthesis of the device T7-LacO Promoter + RiboJ + RBS_0034 + mCherry + double Terminator plus overlaps insert and linear pBBR1MCS-2 plasmids were incubated with Gibson Assembly Master Mix at 50°C for 1 hour. The assembly mixture was then transformed into competent E.coli DH5-alpha cells by heat shock at 42°C for 10 seconds and plated onto Luria Broth Agar plates supplemented with Kanamycin at working concentration for selection. The isolated colonies were cultivated and a miniprep was performed.

A PCR from miniprep plasmid, under the following conditions: 98°C for 40 seconds, 30X cycles of: 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 35 seconds. Final extension at 72°C for 2 minutes. PCR product held at 4°. PCR products were run on a 0,8% agarose gel at 50 V for 45 minutes and visualised in a transilluminator setting. Primers used at the overlap section:

Whole insert Forward :

• 5’-GTGCTGCAAGGCGATTAAGTTG

Whole insert Reverse :

• 5’-TTACAACGTCGTGACTGGGAAC

Figure 3. The first lane is a positive control, with the PCR product from amplification oh this par synthesis (gBlock). A linear sample of pBBR1MCS-2 was used as a negative control. On the third lane, the G0 assembly on E. coli DH5-alpha was confirmed as positive.

Sequence and Features