Difference between revisions of "Part:BBa K3286205"

Revision as of 09:56, 21 October 2019

Vc2 Riboswitch + sfGFP

This composite part contains the Vc2 riboswitch (BBa_K3286202)[1], which can be (and was) used in combination with composite part BBa_K3286204. The function of this part is that upon DSF sensing from part BBa_K3286204, the levels of c-di-GMP will be decreased in the cell. This results in alleviation of the repression of the Vc2 riboswitch[2], which will result in sfGFP (BBa_K3286203) translation.

Figure 1: Comparison of a low copy number(left) and a high copy number (right) plasmid containing this part. Both are E. coli Dh5-Alpha cells incubated on a LB agar plate. Fluorescence is visualized by a blue light transilluminator.

Figure 1 is showing the differences between low and high copy number plasmids transformed with this part. We can can conclude that transformation with the high copy number plasmid pSB1C3 is causing leaky expression of GFP translation, whereas the low copy number plasmid pSEVA62[3,4] is not showing any optical fluorescence. This leaky expression is probably due to the fact that there are too much riboswitch present transcriptions present, outcompeting the c-di-GMP, resulting in GFP translation.

Figure 2: cDNA synthesis from RNA extracted samples. pSEVA23 and pSB1C3 both show one band specific for GFP in triplicate. The positive control also shows one band whereas the negative controls don't show a band. Two colonies and one plasmid were amplified with the GFP specific primers as positive controls

1. B. R. Pursley, M. M. Maiden, M. L. Hsieh, N. L. Fernandez, G. B. Severin, and C. M. Waters, “Cyclic di-GMP regulates TfoY in Vibrio cholerae to control motility by both transcriptional and posttranscriptional mechanisms,” J. Bacteriol., vol. 200, no. 7, pp. 1–19, 2018.
2. S. Inuzuka et al., “Recognition of cyclic-di-GMP by a riboswitch conducts translational repression through masking the ribosome-binding site distant from the aptamer domain,” Genes to Cells, vol. 23, no. 6, pp. 435–447, 2018.
3. R. Silva-Rocha et al., “The Standard European Vector Architecture (SEVA): A coherent platform for the analysis and deployment of complex prokaryotic phenotypes,” Nucleic Acids Res., vol. 41, no. D1, pp. 666–675, 2013.
4. E. Martínez-Garćía, T. Aparicio, A. Goñi-Moreno, S. Fraile, and V. De Lorenzo, “SEVA 2.0: An update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities,” Nucleic Acids Res., vol. 43, no. D1, pp. D1183–D1189, 2015.

Sequence and Features