Part:BBa_K4162027

ribozyme + T7_RBS + ybbO-tdMCP

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.

The short peptide between ybbO and tdMCP acts as a linker which can avoid the conformation of enzymes and tdMCPs interfering with each other. This part can be used to assemble polycistrons in Escherichia coli.

Characterization

HPLC

Figure 1. HPLC. Acetone extracted samples were subjected to HPLC. The extract from bacteria DH5α expressing CAG-MS2, BCMO, ybbO, crtYEBI (the panel above, short name 3-7) compared with the extract from bacteria expressing CAG-MS2, BCMO, ybbO-tdMCP, crtYEBI (the panel below, short name 3td7), there are two peaks in the figure between 2-4 minutes (where our standards appear as well others, detected at 325 nm), the absorbance value of the two peaks of 3td7 in this period is significantly higher than that of 3-7, which depicted the efficiency of TEARS in improving the production of retinol.

Fluorescence microscope image

Figure 2. Fluorescent imaging confirm the expression of both BCMO and ybbO. We observed fluorescence images of E. coli (1. Bacteria expressing BCMO-linker-StayGold ; 2. Bacteria expressing BCMO and ybbO-tdMCP-GFP ) through an Olympus fluorescence microscope with a 1.45 NA 150× oil objective. GFP and StayGold were excited by a 488 nm laser. Images were taken by the same settings for comparison.

Figure 3. Fluorescent imaging revealed that TEARS cluster ybbO in bacteria.We observed fluorescence images of E. coli (Bacteria expressing CAG-MS2, BCMO, ybbO-tdMCP-GFP, crtYEBI) through an Olympus fluorescence microscope. Four different views are shown. Please note for GFP is not required for TEARS to work. During retinol production, we used the bacteria expressing CAG-MS2, BCMO, ybbO-tdMCP-GFP, crtYEBI.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  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