Part:BBa_K1890031
BolA gene with strong constitutive promoter, 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 should become spherical [2]. It is expressed under control of the strong constitutive promoter BBa_J23100, strong RBS BBa_B0030 and terminators BBa_B0010 and BBa_B0012. Our findings are that BolA under a constitutive promoter will result in elongated cells, because of a too high expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 158
- 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. 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. Constitutive expression of the BolA gene, as shown in Figure 1B does not result in spherical cells: the cells are rod-shaped and elongated. This is probably because of the stress the constitutive expression puts on the cells. As mentioned before, BolA is a gene involved in the stress response of E. coli that changes the morphology of the cells. A too high expression of the gene could therefore change the morphology in an unexpected way, such as elongation, a phenomena that is often observed in E. coli [3].
Expression under an inducible promoter usually puts less stress on the cell. Expression of the BolA gene under an inducible promoter did result in spherical cells (BBa_K1890030).
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] Höltje, J.-V. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiology and Molecular Biology Reviews, 62(1), 181-203.
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