Part:BBa_K5136233

J23100-rfbD-B0015
Responsible for expression of rfbD facilitated rhlAB–rhlC to enhance di-rhamnolipids accumulation (1).

Biology

Studies have shown a limited amount of precursors hinder rhamnolipids production (2). So over-expression of rfbD can circumvent the shortage of dTDP-l-rhamnose in E. coli. In our project, the rfbD gene controlled by the constant promoter BBa_J23100 and the RBS BBa_B0034 as well as the terminator BBa_B0015 was assembled into the bacterial expression vector pSB3K3 and transformed into E. coli BL21(DE3).

Usage and Design

We used BBa_K081005 to construct the expression system and obtained the composite BBa_K5136233, which is assembled on the expression vector pSB3K3 by standard assembly (Figure 1). The constructed plasmid was transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Figure 1 Gene circuits of verification system for over-expression of rfbD.

Characterization

1. Agarose Gel Electrophoresis

After transferring the plasmid into E. coli DH5α, colony PCR was used to certify the plasmid was correct. The expected bands(1414 bp) were obtained at the position around 1500 bp (Figure 2).

Figure 2 The result of colony PCR products of BBa_5136233_pSB3K3.

Reference

1.Du, J., A. Zhang, J. a. Hao, J. Wang (2017). "Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered Escherichia coli." Biotechnol. Lett 39(7): 1041-1048.
2.Cabrera-Valladares, N. et al. (2006). "Monorhamnolipids and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) production using Escherichia coli as a heterologous host." Appl. Microbiol. Biotechnol. 73(1): 187-194.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]