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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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