Overview

Why we choose Orthopoxvirus vaccinia Tian Tan（AF095689.1）？

Firstly, vaccinia virus Tian Tan（AF095689.1）is considered the most secure virus within our laboratory setting, having been continuously cultured over an extended period. And we have developed sophisticated and refined methods for the genetic modification of the vaccinia virus, enabling us to tailor its properties for various applications including reserch, vaccine development, and therapeutic uses. It is important to note that, while our modification techniques are highly advanced and effective, their application is specific to the vaccinia virus. These methods do not readily translate to other viral species, due to the unique genetic and structural characteristics that differentiate vaccinia from other viruses. Furthermore, this virus has found substantial application in various domains, notably in vaccine development and as an oncolytic virus for cancer treatment. Importantly, upon infecting a host, vaccinia virus instigates a disease that is self-limiting, meaning it resolves on its own without the need for medical intervention.

In order to conduct further experiments to verify the feasibility of the Click Virus project, the wet lab group carried out two sets of experiments on different themes.

The targets for the two engineering modifications are:

1.Construction of the plasmid pcDNA3.1-6X Myc A27L-mut GFP;

2.Modification of the vaccinia virus to display A27L with a click site on its membrane surface;

3.Verification of the incorporation of non-natural amino acids into A27L.

The entire engineering process is logically executed according to the DBTL (Design, Build, Test, Learn) principle recommended by iGEM. We continue to transform and iterate to improve our project.

This section not only includes our journey of exploration in experimental design and vector construction but also records our continuous improvement in experimental operations. Below is the experimental process and logical network we have completed, for detailed experimental steps, please refer to the Experiment section.

Step1 Modify the virus

Success in the construction of the plasmid pcDNA3.1-A27Lmut-GFP

Research

In the introductory course of chemical biology, we learned about the expansion of the translation system, where scientists incorporate non-natural amino acids into proteins using various methods. Some utilize stop codons, while others use the four-base codon and artificial bases to achieve this goal. Ultimately, we focused on the stop codon UAG to encode 4-L-azidophenylalanine.

After reviewing extensive literature on the structure and properties of the vaccinia virus, our team decided to incorporate 4-L-azidophenylalanine into the membrane protein A27L. A27L affects only the enveloped extracellular virus (EEV) of the vaccinia virus and does not affect the production of intracellular mature virus (IMV). In mammals, only EEV can spread over long distances, while IMV can only spread through cell-to-cell and short-distance transmission. At the same time, A27L is also a key protein for the adhesion of IMV before infecting cells, and its absence in experiments greatly reduces the risk and infectivity, which is very beneficial for our subsequent experiments.

According to the structural characteristics of the A27L protein, its N-terminus is located on the outside of the cell membrane. We used gene-editing techniques to mutate the codons of the three phenylalanines at the N-terminus of A27L into the stop codon AUG. In this way, only by introducing a specific artificial translation system and adding the corresponding 4-azido-L-phenylalanine can the modified vaccinia virus synthesize A27L containing an azide group, which can then connect with specific molecules to achieve the experimental purpose.

Since the phenylalanine codon is changed to the stop codon AUG, the A27L protein cannot be synthesized in full under normal conditions. To allow the mutant vaccinia virus to synthesize a complete A27L, we will introduce an artificial translation system, which includes tRNA that can recognize AUG and aminoacyl-tRNA synthetase (plasmid pcpAzpaRS_v2).

In this way, the engineered click virus can carry A27L with a click site on the membrane surface, allowing it to connect with any target molecule, thereby achieving various purposes such as therapy, diagnosis, and tracing.

Cycle 1

Design

To facilitate the identification and screening of cells containing the mutated virus in subsequent experiments, we plan to insert GFP along with two forward and reverse promoters (which specifically function in vaccinia virus) between the A27L gene and the A28L gene fragment of the viral genome. Based on the ideas and templates provided by the Wuhan Institute of Virology (specific to vaccinia virus), we identified the gene sequence of A27L, introduced mutations after the forward codon, placed the GFP gene fragment after the reverse codon, and then added 500bp homologous arms at both ends of the A27L gene fragment (including the A26L gene) and the GFP gene fragment (including the A28L gene). Under the guidance of our instructor, we modified the A27L gene fragment (as the initially used A27L gene fragment strain differed from the laboratory strain), changed the type of GFP, and adjusted the position of the homologous arms from the end of A27L to the last mutation site on A27L. We then had Qingke Biotechnology Company synthesize this DNA fragment and inserted it into pcDNA3.1-6X Myc. After discussion, we determined that performing homologous recombination to replace the A27L gene fragment would be a better approach. We then identified the A27L gene sequence of the strain we were using and replaced the three base sequences corresponding to phenylalanine at the N-terminus with ATG. Subsequently, we constructed a plasmid that ensures GFP expression only when infected with vaccinia virus. The plasmid includes basic components, the GFP gene, the mutated A27L gene, vaccinia virus-specific promoters, and 500bp homologous arms required for homologous recombination at both ends.

Build

Design a plasmid (pcDNA3.1-6X Myc A27L-mut GFP) with the sequence "homologous arm-GFP-reverse promoter-forward promoter-A27L-mut-homologous arm" (the A27L-mut overlaps with the homologous arm).

The modified VTT A27L gene sequence is as follows:

Figure2.3. the modified VTT A27L gene sequence

Test

After purchasing the plasmid, it is cultured with Escherichia coli DH5-α and the plasmid is extracted.

Learn

The concentration of the extracted plasmid is too low. The analysis indicates that this is because 300 µL of TB was added, whereas only 100 µL should have been added.

Cycle 2

Design

When extracting the plasmid, only 100 µL of TB should be added.

Build

Culture Escherichia coli containing the plasmid pcDNA3.1-A27Lmut-GFP using the streak plate method.

Firuge5.6. the streak plate method was used to cultivate Escherichia coli containing the plasmid pcDNA3.1-A27Lmut-GFP.
Test

Figure7.8.9 The concentration of the plasmid pcDNA3.1-A27Lmut-GFP
Learn

When extracting the plasmid, the amount of TB should be determined based on the quality of the sample. If the density of the cultured colonies is low or the number of plasmids is small, the amount of TB used should be appropriately reduced to increase the concentration of the plasmids extracted.

Cycle 3

Design

Using homologous recombination technology, the vaccinia virus Tian Tan strain (wild type) was made to carry the A27L-mut and GFP sequences, resulting in the vaccinia virus Tian Tan strain (mutant type).

Build

1.Virus infection

2.Transfection with pcDNA3.1-A27Lmut-GFP plasmid

Test

Figure10.11 20240604 293T sham A27L-mut 2.5lipo 10X

Figure12.13 20240604 293T sham A27L-mut 2.5lipo

Figure14.15 20240604 293T VVT A27L-mut 1.5lipo 10x

Figure16.17 20240604 293T VVT A27L-mut 1.5lipo

Learn

The first well without virus solution showed no fluorescence, while the subsequent five wells with virus solution displayed green fluorescent infection spots. This proves that the promoter on the plasmid is a vaccinia virus-specific promoter. The optimal ratio for maximum fluorescence infection spots was found to be plasmid:lipid = 1:2.5.

Step2 Introducing an artificial translation system

Cycle 4

Design & Build

Repeat the cycle of picking clones, infecting, observing, and picking clones until after the sixth infection, under a fluorescence microscope, nearly all plaques display fluorescence.

Test

Figure18. 20240721 A27Lmut 5C2'-4-3-1-2 4X 24hpi

Figure18. 20240721 A27Lmut 5C2'-4-3-1-2 4X 24hpi
Learn

After the sixth infection, nearly all plaques displayed fluorescence under a fluorescence microscope, indicating that the concentration of the modified virus is high and samples with a higher purity of the modified vaccinia virus have been obtained. These can be used for the next step of introducing an artificial translation system to express the modified A27L.

However, fluorescence images only ensure the expression of the GFP protein and do not guarantee that the target gene locus of A27L has been successfully mutated. Therefore, before introducing the artificial translation system, it is necessary to verify whether the target gene locus of A27L has been successfully mutated.

Cycle 5

Design

To verify whether the target locus has been mutated, we designed the following primers:

• A27L-mut-FP: 5’-AACCCTCAAGAACCTTTG-3’ (CG44% Tm:51-52℃)
• A27L-mut-RP1: 5’-TAGCAGCCTTTGTAGACTAC-3’ (CG45% Tm:52-53℃)
• A27L-mut-RP2: 5’-TCGTTGCGTTTACAACAC-3’ (CG44% Tm:54℃)

Figure20. the primer binding sites are as shown in the figure, indicated by red underlines.

As shown in the figure, A27L-mut-RP1 is used to check whether the target gene locus of A27L has mutated, while A27L-mut-RP2 is used for sequencing A27L-mut to ensure that the sequence of A27L-mut is as required.

Build

Perform PCR amplification on the gene segment containing the mutation site of the modified VTT, and conduct agarose gel electrophoresis and analysis on the PCR product.

Test

Figure21. the most left band is the DL 2000 DNA Marker. From left to right, lanes 2-7 are groups 1, 2, 3, 4, 5, and 6 respectively.
Learn

The target product is a 190bp sequence (modified sequence, with the reverse primer A27L-mut-RP2 at the mutation site) and a 300bp sequence (normal sequence).

If the PCR product contains a 300bp sequence, it indicates that the template has undergone homologous recombination, but it does not confirm whether it contains the target mutation site; if the PCR product contains a 190bp sequence, it proves that the template contains the target mutation site. From the imaging results, lane 6 demonstrates that 5C2’-4-3-1-2 has undergone homologous recombination and contains the target gene; lane 6 also proves that 5C2’-4-3-1-2 contains the target mutation site.

In the image, the PCR product around 100bp is a primer dimer. In lane 6, a PCR product of about 250bp can be seen in the middle. Preliminary analysis suggests this could be due to non-specific amplification of PCR or impurities in the virus template. Therefore, the next step is to perform gradient PCR to verify whether the approximately 250bp PCR product is caused by non-specific amplification. From the gel imaging, groups 1, 3, and 5 have double bands, with the lower band being less than 100bp nucleic acid, which is a primer dimer, and the upper band being about 200bp nucleic acid, which is the PCR product, indicating that the target site has mutated. Groups 2, 4, and 6 have lighter bands, and preliminary analysis suggests this is due to a higher annealing temperature. It is necessary to redo the PCR at a Tm of 48℃.

Cycle 6

Design & Build

Reperform the PCR at a Tm of 48℃ and conduct agarose gel electrophoresis and analysis on the PCR product.

Test

Figure22. the most left band is the DL 2000 DNA Marker. From left to right, lanes 2 to 4 are groups 2, 4, and 6 respectively.
Learn

The results indicate that groups 2, 4, and 6 obtained PCR results at a Tm of 48℃. However, there are still double bands, and the preliminary analysis suggests that this is due to the virus not being fully purified. The next day, re-perform the PCR using the upstream primer and downstream primer 1 for verification.

Cycle 7

Design

Verify whether the virus template is fully purified (to verify the cause of the double bands in the previous electrophoresis).

Build

Reperform the PCR using the upstream primer and downstream primer 1 for verification, and conduct agarose gel electrophoresis and analysis on the PCR product.

Test

Figure23. he most left band is the DL 2000 DNA Marker. From left to right, lanes 2 to 4 are groups 2', 4', and 6' respectively.
Learn

The presence of double bands indicates that the virus is impure (this does not exclude the extremely low probability that the primers have additional binding sites).

The increased brightness is due to the fact that the PCR product from the previous round was not purified, resulting in residual primers increasing the total primer concentration.

Tails could be caused by protein impurities.

Success in the virus cloning

Cycle 8

Design & Build

Reperform the infection-observation-pick clones-infection cycle until after the eighth round of infection, when the concentration of the modified virus is higher.

Test
Learn

Preliminary determination indicates that after the eighth round of infection, the concentration of the modified VTT is higher and suitable for PCR reaction.

Cycle 9

Design

Verify whether the virus template is fully purified (to verify the cause of the double bands in the previous electrophoresis).

Build

Conduct PCR for verification using the same primers from the fifth cycle, and perform agarose gel electrophoresis and analysis on the PCR product.

Test

1. From left to right, lanes 1 and 2 are the modified virus templates, lanes 3 and 4 are 5C2’-4-3-1-1, lanes 5 and 6 are 5C2’-4-3-1-2, and lane 7 is the DL 2000 DNA Marker.

2.From left to right, the reverse primer for lanes 1, 3, and 5 is reverse primer 1 (A27L-mut-RP1), and the reverse primer for lanes 2, 4, and 6 is reverse primer 2 (A27L-mut-RP2).

Learn

The target product is a 190bp sequence (modified sequence, with the reverse primer at the mutation site) and a 300bp sequence (normal sequence).

If the PCR product contains a 300bp sequence, it indicates that the template has undergone homologous recombination, but it does not confirm whether it contains the target mutation site; if the PCR product contains a 190bp sequence, it proves that the template contains the target mutation site. From the imaging results, lane 6 demonstrates that 5C2’-4-3-1-2 has undergone homologous recombination and contains the target gene; lane 6 also proves that 5C2’-4-3-1-2 contains the target mutation site.

In the image, the PCR product around 100bp is a primer dimer. In lane 6, a PCR product of about 250bp can be seen in the middle. Preliminary analysis suggests this could be due to non-specific amplification of PCR or impurities in the virus template. Therefore, the next step is to perform gradient PCR to verify whether the approximately 250bp PCR product is caused by non-specific amplification.

Cycle 10

Design

Conduct gradient PCR to verify whether the approximately 250bp PCR product is caused by non-specific amplification.

Build

Perform gradient PCR validation, and analyze the gradient PCR products by agarose gel electrophoresis

Test
Learn

The 6th and 7th lanes of reverse primer 2 (A27L-mut-RP2) show no 250bp product, confirming that there is no non-specific amplification or weak non-specific amplification at 57.6°C and 59.1°C. Therefore, the gradient PCR products from A27L-mut-RP2-6 and A27L-mut-RP2-7 were sequenced to determine whether the target mutation is present in the viral genome.

And the sequencing results confirmed that the gradient PCR products from A27L-mut-RP2-6 and A27L-mut-RP2-7 contain the target mutation.

Success in virus modification

Cycle 11

Design & Build

Sequencing of the gradient PCR products from A27L-mut-RP2-6 and A27L-mut-RP2-7 was conducted to determine whether the target mutation is present within the viral genome.

Test

Figure28. result of sequencing

The sequencing results confirmed that VTT contains three target mutation sites, indicating successful viral modification.

Success in the phagocytic plaque assay

Cycle 12

Design & Build

Crystal violet staining method for calculating virus concentration

Test
Learn

1.Among them, wells 4 and 5 showed better results.

2.Well 4 has 33 plaques; well 5 has 6 plaques.

3.After calculation, the virus stock concentration is 3.67 x 10^5 pfu/mL.

Research

We confirmed the necessary conditions for the incorporation of 4-L-azidophenylalanine into A27L, which require the presence of a bioorthogonal tRNA that can recognize the AUG stop codon and a mutated aminoacyl synthetase that can recognize both this tRNA and 4-L-azidophenylalanine within the host cells. During our literature search, we found a plasmid that meets all three requirements, published in an article by Mr. Wenshe Liu from California Polytechnic State University in 2002. We ordered it through the Addgene platform and then began modifying the viral genome.

Cycle 13

Design & Build

The plasmid pcpAzpaRS_v2 was purchased through the Addgene platform. pcpAzpaRS_v2 can simultaneously transcribe tRNA in mammalian cells (293T) with human modifications at both the 3' and 5' ends, and translate EcTyrRS(Y37L/D182S/F183M/L186A). Additionally, it includes RNA polymerase III.

Figure33. map of plasmid pcpAzpaRS_v2
Test

The plasmid was transformed into competent Escherichia coli DH5α and cultured for 16 hours before plasmid extraction and analysis by agarose gel electrophoresis.

Learn

Cycle 14

Design & Build

The modified virus was used to infect HEK293T cells, introducing a plasmid that encodes the function corresponding to the UAG stop codon (artificial translation system). In this way, the stop codon AUG can code for 4-azido-L-phenylalanine, attaching an azido group to A27L.

Test
Learn

From the fluorescence microscopy images, it can be seen that the purity of the modified virus is high, with almost all plaques caused by the infection of the modified virus.

Success in the construction of the recombinant expression vectors p3×FLAG-CMV-FOLR1-mCherry and p3×FLAG-CMV-FOLR2-mCherry

Cycle 15

Design

Construct the recombinant expression vectors p3×FLAG-CMV-FOLR1-mCherry and p3×FLAG-CMV-FOLR2-mCherry using homologous recombination methods.

Build

1.Purchase plasmids containing FOLR (pLV2-EF1a-FOLR1(human)-IRES-Puro, pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro) to provide the gene sequence of the folate receptor for subsequent plasmid constructions we intend to build.

2.Prepare the laboratory's existing p3×FLAG-CMV-mCherry plasmid, and obtain the mCherry gene fragment for subsequent plasmid construction.

1.Design primers based on homologous recombination methods to insert the folate receptor sequence fragment into the p3×FLAG-CMV-mCherry plasmid.

2.PCR amplify the T2A fragment from the plasmid pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro.

3.Purify the PCR product; measure the extracted DNA content.

4.Digest the p3×FLAG-CMV-mCherry plasmid with restriction enzymes.

5.Purify the digested product; measure the extracted DNA content.

6.Perform homologous recombination:

• 1.Homologously recombine the FOLR1 fragment into the vector p3×FLAG-CMV-mCherry.
• 2.Homologously recombine the FOLR2 fragment into the vector p3×FLAG-CMV-mCherry.

7.Transform the reconstructed expression vector into competent Escherichia coli DH5α.

8.Pick single colonies and shake them in liquid culture.

Test

Extract the plasmids p3×FLAG-CMV-FOLR1-mCherry and p3×FLAG-CMV-FOLR2-mCherry, and measure the DNA content.

Learn

Success in the construction of the recombinant vector p3×FLAG-CMV-FOLR1-T2A-mCherry and recombinant vector p3×FLAG-CMV-FOLR2-T2A-mCherry

Cycle 16

Design

Construct the recombinant expression vectors p3×FLAG-CMV-FOLR1-T2A-mCherry and p3×FLAG-CMV-FOLR2-T2A-mCherry using homologous recombination methods.

We plan to introduce a plasmid that can express a non-green fluorescent protein as well as the folate receptor into 293T cells, in order to achieve subsequent targeted validation (i.e., indirectly verify whether the virus carries an azide group).

Build

1.Prepare the previously constructed recombinant expression vectors p3×FLAG-CMV-FOLR1-mCherry and p3×FLAG-CMV-FOLR2-mCherry plasmids.

2.Design primers based on homologous recombination methods to insert the T2A sequence fragment into p3×FLAG-CMV-FOLR1-mCherry and p3×FLAG-CMV-FOLR2-mCherry plasmids, respectively.

3.PCR amplify the T2A fragment from the plasmid pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro.

4.Purify the PCR product; measure the extracted DNA content.

5.Digest the p3×FLAG-CMV-FOLR1-T2A-mCherry and p3×FLAG-CMV-FOLR2-T2A-mCherry plasmids with restriction enzymes.

6.Purify the digested products; measure the extracted DNA content.

7.Perform homologous recombination:

• Homologously recombine the T2A fragment into the recombinant expression vector p3×FLAG-CMV-FOLR1-mCherry.
• Homologously recombine the T2A fragment into the recombinant expression vector p3×FLAG-CMV-FOLR2-mCherry.

8.Transform the reconstructed expression vectors into competent Escherichia coli DH5α.

9.Pick single colonies and shake them in liquid culture.

Test

Extract the plasmids p3×FLAG-CMV-FOLR1-T2A-mCherry and p3×FLAG-CMV-FOLR2-T2A-mCherry, and measure the DNA content.

Learn

Figure49. result of sequencing the recombinant vector p3×FLAG-CMV-mCherry-FOLR1-T2A

Figure50. result of sequencing the recombinant vector p3×FLAG-CMV-mCherry-FOLR2-T2A
Success in the expression of FOLR1, FOLR2, and mCherry in HEK 293T cells

Cycle 17

Design & Build

Transfect the plasmids into HEK293T cells.

Test
Learn

The plasmid FolR2-T2A-mCherry was successfully transfected and can be used for subsequent validation. The transfection of FolR1-T2A-mCherry failed, and the possible reason may be that the plasmid concentration is too low.

Step3 Using click chemistry to connect viruses with desired molecules
&
Step4 Testing whether the virus has been successfully modified

Success in the virus expressing A27L-3stop & success in the click chemistry reaction between A27L-3stop and folate-PEG-DBCO , resulting viruses with targeted specificity

Cycle 18

Design & Build

The effect of diluting the virus first, then adding folate-DBCO for reaction, and finally transfect.

Test

Figure 53 Fluorescence images of click virus (green) and cells overexpressing FOLR2 (red) under different reactive concentrations of folate-DBCO. The yellow regions in the merge figures indicate the FOLR2-overexpressed cells infected by click virus. The composition column shows the ratios of different colors in each treatment.

Learn

Analysis: As can be seen from the figure, only when 10uM Folate-PEG-DBCO is added do yellow plaques appear, indicating a significantly poor effect.

Cycle 19

Design & Build

First transfect for 12 hours, then dilute the virus and add Folate-PEG-DBCO for reaction, and finally infect the cells.

Test

Figure 54 Fluorescence images of click virus (green) and cells overexpressing FOLR2 (red) under different reactive concentrations of folate-DBCO. The yellow regions in the merge figures indicate the FOLR2-overexpressed cells infected by click virus. The composition column shows the ratios of different colors in each treatment.

Learn

Analysis: As can be seen from the figure, there is a significant amount of yellow resulting from the overlap of red and green fluorescence, indicating a good effect. This confirms that 4-L-azidophenylalanine has been used to synthesize A27L-3stop, resulting in better viral targeting.

References

• [1] Wen, AM and Steinmetz, NF. Design of virus-based nanomaterials for medicine, biotechnology, and energy. Chem Soc Rev. 2016;45(15): 4074–4126.
• [2] Chi, H, Wang, X, et al. Engineering and modification of microbial chassis for systems and synthetic biology. Synth Syst Biotechnol. 2018;4(1):25-33.
• [3] Pokorski, JK and Steinmetz NF. The Art of Engineering Viral Nanoparticles. Mol Pharm. 2011;8(1):29-43.
• [4] Liu, SL, Wang, ZG, et al. Single-Virus Tracking: From Imaging Methodologies to Virological Applications. Chem Rev. 2020;120(3):1936-1979.