Part:BBa_K4768009

Split T7 RNA polymerase (Nterm) conjugated to rapamycin antibody (FRB) with a soluble linker

Part for expression of the Split T7 RNA polymerase (Nterm) conjugated to rapamycin antibody (FRP) with a soluble linker

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 45
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 641
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 45
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 45
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 26
  

Introduction

Figure 1: T7Nterm-SL-FRB structure.

The CALIPSO part BBa_K4768009 is composed of the N-terminal fragment of the T7 RNA polymerase (residues 1 to 180) fused to an anti-rapamycin antibody FRB through a soluble linker (SL). This gene is under transcriptional control of an SP6 promoter and T7 terminator.

This part, coupled to the part BBa_K4768010 containing the C-terminal subunit of the T7 RNA polymerase, has been designed to develop a split T7 RNAP-based biosensor capable of recognizing rapamycin. It was inspired from the article Evolution of a split RNA polymerase as a versatile biosensor platform by Jinyue Pu et al. [1], as shown in Figure 2, who produced the recombinant protein in vivo. The main goal was to produce a functional biosensor with a two-partite RNA polymerase-linked antibody for activity in PURE system.

Figure 2: Functioning of the split T7 RNAP anti-rapamycin biosensor [1].

Construction

The CALIPSO part BBa_K4768009 consists in the N-terminal subunit of the T7 RNA polymerase fused to FRB, an anti-rapamycin antibody, on its C-terminal domain through an 8-amino-acid linker composed of glycine and serine residues.

In order to add an SP6 promoter and an RBS upstream the sequence of interest, as well as a downstream T7 terminator, we ordered two pairs of primers from Eurofins and performed two successive PCR amplification steps.

Figure 3: Gel electrophoresis analysis of the PCR products generated during the two-step amplification reactions. 0.8% agarose gel and EtBr staining were used. (A) Amplification products of the first PCR appeared at the expected size. (B) Amplification products of the second PCR also appeared at the expected size.

Production

The CALIPSO part BBa_K4768009 was first produced using GeneFrontier PUREfrex 2.1, as well as part BBa_K4768010, the second subunit of the biosensor. This kit promotes formation of disulfide bonds in synthesized proteins due to its non reducing environment. We aimed to evaluate the expression of this DNA part in PURE system under these conditions. SP6 RNA polymerase was supplied to the reaction mixture to enable constitutive transcription of the two genes. Moreover, GreenLys reagent was supplemented for co-translational incorporation of fluorescent lysine residues, which facilitated the detection of synthesized proteins by SDS-PAGE. A clear band corresponding to T7Nterm-SL-FRB (32 kDa) was obtained as shown in Figure 4.

Figure 4: SDS-PAGE of the two rapamycin biosensor proteins FKBP-SL-T7Cterm and T7Nterm-SL-FRB expressed in PURE system. DHFR used as positive control (T+) is visible at 18 kDa (lane 3), FKBP–SL-T7Cterm at 91 kDa (lane 4) and Nterm-SL-FRB at 32 kDa (lane 5). The protein marker is in lane 1 and the negative control (T-, no DNA) in lane 2.

Characterization

After validating the production of the two subunits of the rapamycin biosensor in PURE system, we decided to test its activity. In presence of rapamycin, the recombined split T7 RNA polymerase should promote expression of the sfgfp gene that is under control of a T7 promoter.

The activity assay was performed using the PUREfrex 2.1 custom kit devoid of T7 RNAP (present in Solution II of the regular kit) that would otherwise bypass the effect of the synthesized polymerase. GroE chaperones and rapamycin were added in the reaction mixtures. Figure 5 shows that the normalized sfGFP intensity was higher in the presence of rapamycin. This result suggests that GroE enhances protein folding, which enables formation of an active rapamycin-responsive biosensor. More experiments will have to be performed with other chaperones and different concentrations of rapamycin.

Figure 5: Normalized fluorescence intensity of the sfGFP reporter following expression of the rapamycin biosensor with an optimized protocol. Intensity values were normalized using the fluorescence signal of constitutively expressed sfGFP with (green) or without (red) rapamycin.

Conclusion and Perspectives

We have designed a rapamycin biosensor with transcriptional elements that are compatible with expression in PURE system. Cell-free production of the two complementary biosensor proteins was demonstrated. Preliminary experiments suggest that rapamycin-induced formation of an active RNA polymerase from two split fragments is possible when the chaperone GroE assists protein folding. It should be noted that the increase of the sfGFP reporter signal with addition of rapamycin was modest (from 3 to 7%). We encourage future iGEM teams to perform further experiments in order to confirm these results. This construction can be manipulated in a Biosafety level 1 laboratory.

References

1. Pu, J., Zinkus-Boltz, J., Dickinson, B. C. 2017. Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biology 13(4). 432-438.