Part:BBa_K2839017

12.1 Theophylline Riboswitch +sfGFP-luciferase fusion protein

1.Short Description

This part consists of Pupsp1 promoter, the 12.1 Theophylline Riboswitch and the sfGFP fluorescent protein fused with the first 99 nucleotides of luciferase. This part is an improvement of Part:BBa_K784005 and can be used to achieve the desired expression of the marker, depending on the concentration of externally added Theophylline.

2.Biology and Functionality

Aptamers are oligonucleotides or peptide molecules that change their conformation when bound to the target molecule. They can be used for the regulation of gene expression, which becomes dependent of the inducer’s concentration. We use a Theophylline aptamer which achieves translational control of protein production. Gene expression is off when Theophylline is absent, because the RBS and start codon are hidden inside the aptamer’s loop. When theophylline is added, the aptamer’s conformation changes, the RBS is revealed and the ribosomes can initiate translation.

3.Usage in our Project

As we wanted to expand on the TAL Effector stabilized promoters system, we designed a tool that would allow, on top of the stabilization of a promoter, its induction and activation to the desired expression level. As a chassis we used DH5a E.coli cells for hosting the system’s devices. We implemented a theophylline riboswitch, 12.1, which is completely orthogonal and shows high dynamic range, which is essential in order to allow for precise control of the expression level. As a marker, we use a fusion sfGFP protein that consists of the 99 first deoxyribonucleotides of luciferase followed by the sfGFP sequence.

4.Cloning Strategy

The final plasmids containing the desired device consist of three individual parts: A (Pupsp1 promoter fused with 12.1 riboswitch), B (sfGFP marker) and C. Each part’s sequence is flanked by BsaI recognition sites and after digestion with BsaI restriction enzyme, part A has sticky ends with part B and part C, while part B has sticky ends with part A and part C.In order to successfully clone the Riboswitch 12.1 fusion sfGFP cassette into pSB1C3 vector we followed this procedure:

• PCR amplification of psb1c3 vector with a set of standardized primers.

These primers amplify the plasmid backbone without the insert, incorporate BsaI sites at prefix and suffix, forming the amplified pSB1C3 vector (part C).

• Design and synthesis of the part A, part B fragments with the appropriate flanking regions.
• Golden Gate assembly between part A, part B and the amplified pSB1C3 Vector (part C). This reaction forms the final plasmid Ribo12.1-pSB1C3 containing the riboswitch characterization device.

5.Part:BBa_K784005 Improvement and Characterization

This part is an improvement of BBa_K784005. According to the experimental data submitted by iGEM12_Technion Team the theophylline riboswitch they used did not respond to changes in theophylline concentration. We concluded that the lack of functionality stemmed from a disturbance in the riboswitch’s conformation. The RNA transcript of the fluorescent marker mCherry, which they used could have caused changes in its secondary structure.

In order to assess its functionality, we, initially, used sfGFP as a reporter, but once again no fluorescence was detected. In order to address this issue, we modified the N-terminal reporter region in order to avoid undesired changes in the riboswitch secondary structure. Specifically, we used sfGFP fused with the first 99 nucleotides of luciferase CDS in the 5’ UTR. We selected sfGFP over other available marker since its maturation and fluorescence is unaffected by fusion partner misfolding.

Characterization

a)Sample preparation

In order to prepare the cultures for flow cytometry analysis we followed the protocol created by Adam Mayer et al [1]. In particular the correct colonies were inoculated into 1 ml Lb + antibiotics and grown overnight at 37 °C in a shaking incubator adjusted to 250 rpm.The overnight growths were diluted 1:200 into 1 ml LB + antibiotics and grown at 37 °C into shaking incubator .After 2 hours the growths were diluted 1:500 into prewarmed LB + antibiotics + inducer where necessary and grown at 37 °C, 250 rpm for 5 hours.After growth, 20 μl of culture sample was diluted into 180 μl PBS + 200 μg/ml kanamycin to inhibit translation. The samples were stored at 4°C for 1 hour and then measurements were performed using the CyFlow Cube8 Sysmex Partec Flow Cytometer.

a)Measurement

Fig1: Performance of the two different constructs in response to different theophylline concentrations. The error bars represenr standard deviation from 3 biological replicates.

We restored BBa_K784005 functionality using N-terminal Fusion sfGFP with the first 99 nucleotides of luciferase.

7.References

[1]A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Research, 37(1), 184–192. https://doi.org/10.1093/nar/gkn924

[2]Espah Borujeni, A., Mishler, D. M., Wang, J., Huso, W., & Salis, H. M. (2016). Automated physics-based design of synthetic riboswitches from diverse RNA aptamers. Nucleic Acids Research, 44(1), 1–13. https://doi.org/10.1093/nar/gkv1289

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 802
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 220
  
• 1000
  COMPATIBLE WITH RFC[1000]