Part:BBa_K4162016

ribozyme + T7_RBS + crtI

Introduction

Coding sequence of crtI under hammerhead ribozyme, with T7 ribosome binding site BBa_K4162006 between them. This part can be used to assemble polycistrons in Escherichia coli.

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 1～6: pET28 plasmids encoding crtI，BCMO without any tag were transformed into BL21(DE3) HI-Control strain, single clones (I3, BCMO1, BCMO2) were picked for liquid LB culture. Lane 8~17: pET28 plasmids encoding crtI，crtB, crtE，crtY without any tag were transformed into BL21(DE3) HI-Control strain, single clones (I6, I7, B1, E1, Y1) 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 (4~20% gradient gel, Tanon brand). 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 1~4, 6~9, 11~17: pET28 plasmids encoding crtYB, IB, EY separated by self-cleaving ribozyme, crtB, crtE, crtY without any tag were transformed into BL21(DE3) HI-Control strain, single clones (1YB1/IB7/IB8/1B1/EY3/EY4/1E1/1Y1) were picked for liquid LB culture. Lane 10: pET28 plasmids encoding crtI were transformed into BL21(DE3) HI-control strain, single clones (1I7) 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 (4~20% gradient gel, Tanon brand).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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]