Part:BBa_K4162109

ribozyme+RBS+CDS module: crtIEB

Introduction

This biobrick was created through overlapping assembly of Part:BBa_K4162016 (ribozyme+RBS+crtI), Part:BBa_K4162010 (ribozyme+RBS+crtE) and Part:BBa_K4162013 (ribozyme+RBS+crtB). These genes are a part of the carotenoid biosynthesis pathway.

Characterization

SDS-PAGE

Figure 1. SDS-PAGE. IPTG(-/+) = without/with 0.2 mM IPTG for 3-6 hours, adding IPTG to a bacteria culture with OD600 0.2-0.3. M: Protein molecular weight marker ladder. Lane 2～15: pET28 plasmids encoding crtIEB separated by self-cleaving ribozyme, crtB+crtE+crtY Part:BBa_K4162110, crtB+crtE Part:BBa_K4162103 without any tag were transformed into BL21(DE3) HI-Control strain, single clones were picked for liquid LB culture. Protein expression was induced in parallel cultures by IPTG. Bacterial cultures were monitored by OD600, and 5x10^7 cells were harvested by centrifugation and lysis in 1x SDS sample buffer. Equal amount (10 μL, 2x10^6 cells) of whole cell lysate were analysed by SDS-PAGE (14% separation gel). Red arrows point to crtI protein. Green arrows point to crtY protein. Black arrows point to crtB protein. Yellow arrows point to crtE protein.

Figure 2. SDS-PAGE. IPTG(-/+) = without/with 0.2 mM IPTG for 3-6 hours, adding IPTG to a bacteria culture with OD600 0.2-0.3. M: Protein molecular weight marker ladder. Lane 2～10、13～15: pET28 plasmids encoding crtIEB separated by self-cleaving ribozyme, crtB+crtE+crtY Part:BBa_K4162110, crtB+crtE Part:BBa_K4162103 without any tag were transformed into BL21(DE3) HI-Control strain, single clones were picked for liquid LB culture. Lane 11~12: pET28 plasmids encoding crtBE separated by self-cleaving ribozyme without any tag were transformed into BL21(DE3) HI-Control strain, single clones (BE13) were picked for liquid LB culture. Protein expression was induced in parallel cultures by IPTG. Bacterial cultures were monitored by OD600, and 5x10^7 cells were harvested by centrifugation and lysis in 1x SDS sample buffer. Equal amount (10 μL, 2x10^6 cells) of whole cell lysate were analyzed by SDS-PAGE (14% separation gel).Red arrows point to crtI protein. Green arrows point to crtY protein. Black arrows point to crtB protein. Yellow arrows point to crtE protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 152
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3201
  Illegal NgoMIV site found at 3331
  Illegal AgeI site found at 2359
  
• 1000
  COMPATIBLE WITH RFC[1000]