Part:BBa_K1141002:Design
Plac-RBS-KillerRed (IPTG-inducible)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 133
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 289
Illegal BsaI.rc site found at 580
Design Notes
In the original sequence coming from the pQE30 plasmid (see part BBa_K1141001) there was an Ecor1 restriction site between the promoter and the RBS. We removed this site in order to made this biobrick compatible with RFC[10], and 3A assembly.
Construction
The KillerRed gene that we obtained initially was in an eukaryotic plasmid. To express KR in E. coli and characterize its effects in response to light stimulations, we decided to clone KR into the commercial prokaryotic expression vector pQE30 (Qiagen). This plasmid contains a pLac promoter and a Shine-Dalgarno Ribosome Binding Site (RBS) that allow gene expression in response to the presence of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The pLac-RBS-KR sequence in the pQE30 vector has been submitted as BBa_K1141001. Furthermore, the pLac-RBS-KR sequence was cloned into the pSB1C3 plasmid, to give the biobrick BBa_K1141002. Both BBa_K1141001 and BBa_K1141002 were sent to the standard registry part (Fig 3.).
The choice of an inducible promoter is linked to the absence of literature about the effects of KR on cells in low light. Since KR could be cytotoxic and prevent bacteria from growing even at low doses of light, we wanted to be able to control its intracellular concentration. A negative control for KR characterization was also required. We decided to use the fluorescent protein mCherry, which displays the same excitation and emission spectra as KillerRed [4], and was shown not to be cytotoxic upon light illumination [5]. pSB1C3::pLac-RBS-mCherry (BBa_K1141000) was thus constructed from the existing biobricks BBa_R0010 and BBa_J06702 (Fig 3.).
Figure 3.
Biobricks BBa_K1141002 (A) and BBa_K1141000 (B) used for characterizing KR. C. Picture of KR-expressing bacteria.
Source
The KillerRed coding sequence originally came from the ADDGENE plasmid pBabe-KR-CENPA