Part:BBa_K4162028

ribozyme + T7_RBS + ybbO-tdMCP-EGFP

Introduction

Gene ybbO tagged with tdMCP can be combined with the stem ring structure of MS2, so that retinol dehydrogenase expressed by gene ybbO can be enriched in the area of TEARS. This part can be used to assemble polycistrons in E. 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 10~17: pET28 plasmids encoding crtYEB separated by self-cleaving ribozyme, crtY, crtE, crtB without any tag were transformed into BL21(DE3) HI-Control strain, single clones(YEBd, YEBe, 1Y1, 1E1, 1B1)were picked for liquid LB culture. Lane 1~2, 4~8: pET28 plasmids encoding ybbO-tdMCP-EGFP, BCMO-ybbO-tdMCP-EGFP were transformed into BL21(DE3) HI-Control strain, single clones(2YtG15, 2BY14tG14)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. Purple arrows point to ybbO protein.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1612
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1612
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1612
  Illegal AgeI site found at 109
  Illegal AgeI site found at 702
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1192
  Illegal BsaI site found at 1546