Part:BBa_K5335003

MS2 coat protein virus-like particle loading system

The part is composed of BBa_K5335000, BBa_K5335001, BBa_K5335002, promoter J23110, J23119, rrnB T1 terminator, and ribosome binding site B0034. The whole circuit can play the role of VLP assembly, surface display of functional proteins, and the inner inclusion of functional RNA.

Expected function and circuit construction

Introduction

Our project "Bacillus Gemini" aims to improve plant immunity while killing nematodes. After a large number of previous literature review, we chose Virus-like particles (VLPs) composed of coat proteins of bacteriophage MS2 (MS2 CP) of Escherichia coli as the platform for executive functions, and displayed insecticidal proteins and plant immune activation proteins on its surface to complete the role of killing nematodes that damage plants and activating plant immunity. At the same time, we also included a dual tandem 19bp stem-loop sequence that can be specifically bound by the MS2 coat protein to verify its loading capacity as a future RNA delivery vector. We plan to make MS2 coat protein express in the engineered bacteria and form VLP, bind to the functional protein with SpyCatcher on the surface, and enclose the RNA containing a 19-bp sequence of stem rings to form a multifunctional carrier (Figure 1.).

Figure 1. VLP delivery system.

The plasmid vector was successfully constructed by homologous recombination

Our designed coat protein was constitutively expressed in the engineered bacteria. To obtain sufficient amounts of VLP, we chose to fuse the MS2 CP coding sequence to a high-copy PUC57 mini plasmid. Meanwhile, SpyCatcher-EGFP coding sequence and a 19-bp tandem stem-loop sequence were ligated downstream of the MS2 CP coding sequence (Figure 2.). Upstream of SpyCatcher, we have reserved a place to integrate Psal. Upstream of MS2 CP, we have reserved a place for access to the salicylic acid response element NahR.

Figure 2. Composite PUC57 mini plasmid vector.

We plan to verify the connection function of induced SpyCatcher and the function of loaded RNA after the successful verification of salicylic acid regulatory elements. After homologous recombination, we obtained the target plasmid vector (Figure 3.).

Figure 3. Colony PCR and sequencing confirmed the successful assembly.

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
  Illegal NheI site found at 2317
  Illegal NheI site found at 2340
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 392
  Illegal BsaI site found at 821
  Illegal BsaI site found at 1389
  Illegal BsaI.rc site found at 2096