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-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 pBbA5c-RFP, a gift from Jay Keasling (Addgene plasmid # 35281) [3].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 125
- 1000COMPATIBLE 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.
In Figure 1A we can see cells with the characteristic wildtype E. coli phenotype; the cells are rod-shaped. Figure 1B 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.
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:
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 inducible 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
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