Difference between revisions of "Part:BBa K5335003"

Revision as of 20:16, 30 September 2024

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.

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

Sequence and Features

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 expected molecular weight of SpyCather-EGFP containing 6x His tag and the complex based on SpyCatcher-SpyTag connection were 47.4 kDa and 76.5 kDa, respectively. The result is shown in Figure 4.

Figure 4. Experimental results of protein extraction

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. Purification results of the products expressed at 12 h. Line 1: supernatant. Line 2: BL21 (DE3) Line 3: Engineered bacteria introduced into the plasmid. Line 4 and 6: Purified sample at 12 h. Line 5: precipitate. Red boxes indicate bands that may be target proteins.
C. Purification results of the products expressed at 16 h. Line 1: supernatant. Line 2: precipitate. Line 3: BL21 (DE3). Line 4: Engineered bacteria introduced into the plasmid. Line 5 and 6: Purified sample at 16 h. Red arrows indicate bands that may be target proteins.
D. Comparison of the concentration of the same volume protein purified solution at 12 h and 16 h. Each group selected the same batch of the same volume of three bottles of bacterial culture solution, respectively purified, the same volume of purified solution to determine the protein concentration. "**" indicates p < 0.01. MW is short for Molecular weight.

Through the previous exploration of expression conditions, we found that longer low temperature culture helped to increase the content of purified proteins containing 6x His tag. Subsequently, we controlled the time of low temperature expression at 32 h for expression and subsequent verification. The culture steps are the same as before, but the culture time at 4℃ and 160 rpm is changed to 32 h, and the culture volume is expanded to 200 mL. The process of breaking up the bacteria and purifying the protein containing 6x His tag is also the same as before. Since no purified tag is present on the MS2 CP-Spytag protein, we can only prove the presence of MS2 CP by utilizing 6x His tag on Spycatch-EGFP after successful covalency of the SpyTag-SpyCatcher system. The expected molecular weight of SpyCather-EGFP containing 6x His tag and the complex based on SpyCatcher-SpyTag connection were 47.4 kDa and 76.5 kDa, respectively. We conducted SDS-PAGE on the protein components of the broken cells of the engineering bacteria and the purified protein components, and further conducted Western Blot analysis on the purified components, successfully proved the existence of the target components and the correct connection of the SpyTag-SpyCatcher system (Figure 5.).

Figure 5. SDS-PAGE and Western Blot of target protein. a and b are the eluents with the second highest protein concentration and the highest protein concentration in the purification process.
A. SDS-PAGE of target protein. 1. Impurity protein eluent a. 2. Impurity protein eluent b. 3. Target protein eluent a. 4. BL21 (DE3) strain. 5. supernatant. 6. precipitate. 7. Target protein eluent b. MW: Molecular weight.
B. Western Blot of target protein. 1.Target protein eluent a. 2.Target protein eluent b. MW: Molecular weight.

Preliminary validation of RNA packaging

In our project design, we plan to use this VLP as a multi-functional delivery platform. Therefore, in addition to its ability to display functional proteins on the surface, it also has the function of containing RNA with a specific 19 bp stem-loop structure . In the process of verification, we preliminarily demonstrated some interaction between RNA and MS2 CP through a simple gel retardation analysis.

Gel retardation analysis

The stem loop structure can bind specifically to MS2 coat protein and is encapsulated in VLP. We plan to use this as an anchor sequence for the inner envelope RNA in the future, so we preliminarily verified its binding activity by this assay. By reviewing the literature, we refer to a method for the preliminary detection of protein binding to nucleic acid. It has been documented in the literature that MS2 VLP can be completely eluted during the Ni-NTA column purification process^[1].
We mixed the purified sample which is expressed at 32 h 1:1 with TNE buffer (V/V) and then mixed it with DNA loading buffer containing glycerol for electrophoresis in 1% agarose gel. After electrophoresis, the gel was stained with 1:10,000 dilute safe red dye solution, and the results were observed and recorded. The gel was then stained with G250 dye for protein components, and the strip location was recorded after decolorization with decolorization solution. The results showed that the chromogenic site of nucleic acid was the same as that of protein and its position should be much longer than its own length, which could preliminatively indicate the existence of RNA and coat protein, so that RNA was not hydrolyzed by enzyme

Figure 6. Gel retardation analysis different staining conditions. The left image is the result after dyeing with Safe Red dye, and the right image is the result after dyeing with G250 dye. Line 1: DNA loading buffer. Line 2: DNA loading buffer + TNE buffer. Line 3: Purified protein + TNE buffer + DNA/RNAase + DNA loading buffer. Line 4: DNA loading buffer + TNE buffer + DNA/RNAase

VLP assembly verification

Purification of VLP particles by density gradient centrifugation

Observing the assembled morphology of the VLP requires the use of transmission electron microscopy, which means that we need to prepare high concentrations of the initial protein for density gradient centrifugation. Therefore, we cultured 1 L of bacterial solution according to the previous 32 h low temperature expression method in order to increase the concentration of soluble protein in the crushed supernatant. Due to the large amount of culture this time, we changed the subsequent methods of protein extraction and protein enrichment.The protocol for protein extraction and enrichment this time was as follows.

1). The low-temperature culture products were placed at 4℃ and centrifuged at 6000 rpm for 30 minutes to collect bacteria.

2). The collected bacteria were re-suspended with 50 mL of PBS buffer. The protease inhibitor PMSF was added.

3). The pre-treated samples were then beaten with an ultra-low temperature high-pressure cell crusher at a pressure of 1000bar until the resuspended bacterial solution was no longer viscous.

4.)Centrifuge the crushing liquid at 12000 rpm at 4℃ for 30 minutes, separate the supernatant and precipitate, and store them at 4℃ respectively.

5). 3.7 M ammonium sulfate was mixed 1:1 with the supernatant and precipitated for 12 h at 4℃. The precipitate was subsequently removed and collected by centrifugation at 11000xg for 30 minutes. The precipitate was resuspended in PBS buffer and stored at 4℃.

6). The resuspension was filtered once using a filter membrane with a pore size of 0.22 μm to remove large impurity particles.

The samples prepared after the above steps can be used for density gradient centrifugation and the VLP particle fractions can be easily separated.

We performed ioxanol density gradient centrifugation to separate possible VLP components in the samples. In the experiment, we prepared 7 mL of iodixanol solution in four gradients of 15%, 25%, 40% and 50% at a ratio of 1:3:2:1, and added 5 mL of sample to each tube. Samples were centrifuged at 36000 rpm for 4 h at 4℃. The last bands showing separation lie between a 25% and 40% gradient (Figure 7.).

VLP assembly was observed by transmission electron microscopy

The strips obtained by density gradient centrifugation were re-suspended and diluted 1:1 with PBS buffer. The diluted protein solution is dropped on the copper mesh for 30 seconds to settle fully, and then drained with filter paper sheets after 30 seconds. Then the copper mesh is stained with negative dye for 30 seconds and the dye is sucked up. Finally, the dyed copper mesh was placed on the scaffold and observed by transmission electron microscope. As a result, we observed the assembled VLP particles, and the attached surface display proteins were visible on the particles (Figure 8 B and Figure 8 C).

Figure 8. Density gradient centrifugation results and correct assembly of VLP particles.