Difference between revisions of "Part:BBa K4768009"
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<h2>Production</h2>
 
<h2>Production</h2>
 
<p>The CALIPSO part BBa_K47680009 was first produced using PUREsystem 2.1. 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 FRB-T7Nterm (32 kDa) was obtained as shown in Figure 3.</p>
 
<p>The CALIPSO part BBa_K47680009 was first produced using PUREsystem 2.1. 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 FRB-T7Nterm (32 kDa) was obtained as shown in Figure 3.</p>
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            <figcaption class="normal"><span class="titre-image"><i><b>Figure 2: SDS-PAGE of the two rapamycin biosensor proteins FKBP-SL-T7Cterm and T7Nterm-SL-FRB expressed in PURE system.</b> DHFR used as positive control is visible at 18 kDa (lane 3), FKBP–SL-T7Cterm at 91 kDa (lane 4) and FRB–SL-T7Nterm at 32 kDa (lane 5). The protein marker is in lane 1 and the negative control (no DNA) in lane 2.</i></span></figcaption>
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Revision as of 08:25, 8 October 2023


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: NT7-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], who produced the recombinant protein in vivo. Our main goal was to produce a functional biosensor with a two-partite RNA polymerase-linked antibody for activity in PURE system.

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 2: 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 appeared also at the expected size.

Production

The CALIPSO part BBa_K47680009 was first produced using PUREsystem 2.1. 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 FRB-T7Nterm (32 kDa) was obtained as shown in Figure 3.

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

Characterisation

TXXXXXX.

Conclusion and Perspectives

TXXXXXX.

References

  1. article 1 xxxxxxxx
  2. article 2 xxxxxxx
    3.