Part:BBa_K1890030

BolA gene with RBS and terminator

Introduction

BolA is a gene that controls the morphology of Escherichia coli in the stress response [1]. By overexpressing this gene at 37°C, naturally rod-shapedE. colicells 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) [3].

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. This was tested using two types of imaging techniques: widefield microscopy and scanning electron microscopy (SEM).

Widefield microscopy

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 with BolA under the inducible Lac-promoter indeed has changed the phenotype of the cell. The cells have clearly become spherical

Scanning electron microscopy

To get a more detailed view of the shape of the cells transformed with BolA, we also imaged the cells using SEM, the results are shown in Figure 2.

Figure 2: SEM images of (A) E. coli BL21 without the BolA gene, (B) E. coli transformed with the BolA gene.

In figure 4, we see that the cell shape of the cells transformed with BolA is indeed more spherical than the wildtype phenotype of E. coli. Also, we see that the size of the cells increased. Using ImageJ we analyzed the average size of 10 cells:

Table 1: Average sizes of a population of untransformed E.coli and E. coli transformed with BolA. We measured 10 cells for ech case, the size was determined using ImageJ.

Phenotype	Average length (µm)	Average diameter (µm)
Wildtype	1.2 +/- 0.12	0.5 +/-0.04
BolA	1.0 +/- 0.15	0.87 +/- 0.13

Concluding, we successfully altered the shape of E. coli form rod shaped to spherical, using this biobrick under a constitutive promoter.

References

[1] Santos, J. M., Freire, P., Vicente, M., & Arraiano, C. M. (1999). The stationary‐phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Molecular microbiology, 32(4), 789-798.

[2] Aldea, M., Hernandez-Chico, C., De La Campa, A., Kushner, S., & Vicente, M. (1988). Identification, cloning, and expression of bolA, an ftsZ-dependent morphogene of Escherichia coli. Journal of bacteriology, 170(11), 5169-5176.

[3] Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., … Keasling, J. D. (2011). BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Journal of Biological Engineering, 5, 12. http://doi.org/10.1186/1754-1611-5-12