Part:BBa_K3286205

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 pSEVA62 [3,4] (left) and a high copy number pSB1C3 (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 conclude that transformation with the high copy number plasmid pSB1C3 is causing leaky expression of GFP translation, whereas the low copy number plasmid pSEVA62 is not showing any optical fluorescence. This leaky expression is probably due to the fact that there are too much riboswitch transcriptions present, outcompeting the c-di-GMP, resulting in GFP translation.

Figure 2: Amplification of the cDNA with GFP specific primers. The ladder that was used was a 1 Kb+ ladder from NEB. The expected band should be 207 bp. (1-3) pSEVA23 1 Plasmid system. (4-6) pSB1C3 samples with the Vc2 riboswitch + GFP. (7-9) Positive control contained a constitutively expressed GFP. (10-12) Negative control, which is an E. coli Dh5Alpha strain. (13,14) Two positive colony PCR controls with a transformed GFP sequence. (15) Negative colony PCR control. (16) Positive control on an extracted plasmid containing GFP.

In figure 2 the cDNA synthesis of the RNA extracted samples is shown. This cDNA was used as a template in PCR by amplifying GFP with specific primers for this gene. The results from figure 6 indicate that there is cDNA present with transcribed GFP from both the systems, also the positive control showed a band. The negative control showed no GFP presence. The optical fluorescence of this composite part can be visualized in figure 1.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 392
  
• 1000
  COMPATIBLE WITH RFC[1000]