Part:BBa_K1890030

BolA gene with RBS and terminator

Introduction

BolA is a gene that controls the morphology of E. coli in the stress response [1]. By overexpressing this gene at 37°C, naturally rod-shaped E. coli cells will become spherical [2]. This parts consists of coding sequence, strong RBS BBa_B0030 and terminators BBa_B0010 and BBa_B0012.

This biobrick was expressed under the control of an inducible promoter (Lac-promoter), to do so it was cloned in a backbone containing the promoter and all machinery necessary for it to work. This backbone was obtained from pBbS5a-RFP, a gift from Jay Keasling (Addgene plasmid # 35283) [4].

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 NgoMIV site found at 125
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

This part was expressed in E. coli BL21. The expected phenotype of the cells expressing BolA is very different from the phenotype of wildtype E. coli. If the gene is successfully overexpressed, the cells become round, which we can easily observe under a widefield microscope (Figure 1). In widefield microscopy, the whole sample is simultaneously illuminated using a white light source so the phenotype of the sample can be inspected. This is comparable to normal light microscopy.

Figure 1: Widefield images of E. coli BL21 transformed with a gene not altering the cell shape (K1890002) (A) and with this part (B).

In Figure 1A we can see cells with the characteristic wildtype E. coli phenotype; the cells are rod-shaped. Figure 2B shows that induction of the cells transformed wil BolA under the inducible Lac-promoter indeed has changed the phenotype of the cell. The cells have clearly become spherical