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.).
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE 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 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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
Successful validation of protein expression
Bacterial culture and protein extraction
We preserved BL21 (DE3) strains that had previously verified the correct sequence introduction. We first explored the effect of different time periods of low temperature expression on the concentration of the same volume of purified 6x His tag protein. We cultured the same 25 mL LB medium at different temperatures. The cultivation conditions are as follows.
(1). LB medium containing 60 μg·mL-1 ampicillin was added with 100 μL of preserved bacterial solution, and cultured in a shaking bed at 37℃ at 200 rpm until OD600 was between 0.6-0.8.
(2). The culture with OD600 meeting the requirement was removed and transferred to a shaking bed at 16℃ and 160 rpm for low temperature expression for 12 h and 16 h, respectively.
(3). The low-temperature culture products were placed at 4℃ and centrifuged at 6000 rpm for 30 minutes to collect bacteria.
(4). The collected bacteria were re-suspended with 8 mL of PBS buffer, broken for 10 s using an ultrasonic cell crusher, and broken for 10 s for 30 minutes. Centrifuge the crushing liquid at 12000 rpm at 4℃ for 30 minutes, separate the supernatant and precipitate, and store them at 4℃ respectively.
(5). All the supernatants were purified with Ni-NTA purification kit, and the concentration of purified samples with different expression time was determined, and SDS-PAGE experiment was performed.
The result is shown in Figure 4
A. SDS-PAGE of total protein sample after 12 h expression. Line 1: supernatant. Line 2: precipitate. Line 3: BL21 (DE3) Line 4: Engineered bacteria introduced into the plasmid.B. C. D.
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