Engineering success for the development of an edible vaccine
Our project aims to use synthetic biology to engineer an edible vaccine against SARS-CoV-2 virus. Our engineering success and project design work was parallel and that’s why we chose to present these two as mentioned below. We divided our project in modules and using the Research → Imagine → Design → Build → Test → Learn → Improve → Research design cycle, we decided to set up a series of lab tests to see how effective a vaccine strategy like the one we suggest can be.
Module 1: Theoretical experiment and design
From November 2020 to mid-July 2021 Greece was under general lockdown. Because we were unable to enter a laboratory, we took advantage of the situation by conducting research and compiling a bibliography to build our project's principal idea. We examined the mechanism of mucosal immunity under the direction of Dr. Spilianakis, while Dr. Sarris lectured us on bacterial transformation and agroinfiltration.
Module 2: Golden Gate Cloning
We wanted to construct our own artificial three-dimensional spike protein to cover three Covid 19 strains (the most common ones at the time): the wild type, the Alpha mutation (B.1.1.7) originally discovered in the UK, and the Beta mutation (B.1.351) first discovered in South Africa. Through our research, we looked into the cloning process to find the best way to swiftly and easily insert fragments into our vector. At the same time, we wanted the cloning method we found to require as few enzymes as possible while still ensuring that the fragments were inserted in a certain order so that they could be transformed into a properly folded protein later. Following our investigation, we concluded that Golden Gate Cloning is the best technique for our project since it fulfills all of the aforementioned criteria.
When a pair of classical type 2 restriction enzymes are used to release a desired piece of DNA from a plasmid, some of the recognition sequence depicted in orange will be present at the ends of the liberated fragment. This implies it can only be joined to other pieces of DNA with compatible ends, which are usually those cut with the same enzyme. When this happens, restriction enzyme recognition sequences will be present between the two fragments which are known as assembly scars. Golden Gate cloning uses type 2 restriction enzymes which cleave outside of their recognition sequence. The recognition sequence for BbpI one, for example, is GAAGAC (see figure below), however the cut occurs six base pairs downstream of the recognition sequence, leaving base 5' prime overhangs.
The identification of bases in the cleavage site is not required by sequence. In this example, a blue DNA fragment is flanked in opposite orientations by two Bbsl recognition sequences.
Design and Ordering Primers for the Golden Gate Cloning Assembly
Finding the sequence: First find the sequence of the gene we want to amplify in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (https://www.kegg.jp/)
Finding the sequence of primers: primers must be at least 17 nucleotides long. For the 5' end of the primers, copy the 5' end of the gene coding sequence and for the 3' end, copy the 3' end and obtain the reverse complement sequence (https://www.bioinformatics.org/sms/rev_comp.html). We then use the NEB Tm calculator (https://tmcalculator.neb.com/#!/main) in which we put the primers, and check the deconvolution temperature (Tm). We make sure that the primers between them have no difference in Tm greater than 3.
Modify the sequence: In case overhangs are to be added to the sequence (e.g. for Golden Gate) they are added now.
Checking the interactions of primers with the sequence and with each other: In DNAMAN we can see where each primer binds to the sequence (Load primer, Complementarity on DNA), whether it creates loops and hairpins (Self-complementarity), and the interactions between primers (Two primer complementarity). In the latter case we are interested in the absence of interactions at the 3' ends of the primers.
Control of expected parts after digestions: We check the final sequences for recognition by specific restriction enzymes (e.g. BsaI) in the Vector NTI application or on the Benchling website (https://www.benchling.com/).
Confirm the specialty of the primers: open the blastn website (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) and enter one or both primers (if the system accepts them). We select the organism we want, activate the "Somewhat similar sequences (blastn)" option, and the "Show results in a new window" option. We copy the coordinates of the range, click on the Sequence ID, and by searching for the coordinates (Ctrl+F) in the web page that opens, we check if they refer to the gene of interest (we can also verify the expected size of the PCR product).
Order form: We make an excel file where we put the following columns in the following order: gene name_Fw, Forward primer, Length, Gene name_Rv, Reverse primer, Length, Tm, Length (bp), Comments. We send the form to LabSupplies Scientific.
When these enzymes are used to release a piece of DNA from a plasmid, no part of the recognition sequence is present in the liberated fragment. It can be ligated to other DNA fragments with compatible ends, and no restriction enzyme recognition sequences will be present when the two pieces of DNA are brought together with a DNA ligase.
The most common method of Golden Gate Cloning is an all-in-one reaction. This means that all of the DNA components, as well as the type II restriction enzyme and ligase, are combined in a PCR tube and placed in a thermocycler. The DNA sections are digested and ligated over and over again by cycling between 37°C and 20°C 10 to 50 times. Digested DNA fragments are either religated into their plasmids or combined with additional components as previously described. The assembled pieces become "stuck" in the intended construct because they lack restriction sites for the type IIs enzyme. This is why Golden Gate Cloning has such a high success rate in assembling DNA fragments.
Module 3: Chimeric protein expression
Synthetic protein modeling
We created a model to ensure that the synthetic protein we wish to manufacture is correctly trimerized and folded so that it can be recognized by immune system M cells. We observed the predicted outcomes while using the program AlphaFold2 neural network-based model3 , which allowed us to continue with the design. You can check our modelling process and results here.
Synthesis of our Chimeric protein
Transmembrane expression:To date, all edible vaccines have expressed the protein intracellularly in the hopes of the cell breaking down in the stomach and the protein leaking out extracellularly. This is a fundamental distinction between our effort and previous edible vaccination initiatives. After our meeting sessions with Dr. Zakinthinos and Dr. Papachatzis, and parallel to our research, we chose to go one step further and build our edible vaccine in such a way that the protein gets transported to the cell membrane. We did this to optimize the vaccine's efficiency by ensuring that the protein in the membrane will be identified by immune system cells without the necessity for cell breakdown, resulting in the immunization of the intestinal epithelium. Furthermore, because more proteolysis occurs within the cell than at the membrane, transmembrane protein ensures greater stabilization.
After a few days of deciding that our research would be an edible vaccination, the biggest issue our team encountered was determining the structure of our chimeric protein. The first thing that came to our minds was the transmembrane receptor kinases.After a few days of research, we eventually ended up being inspired by the FLAGELLIN SENSING 2 (FLS2) receptor.
Choosing a suitable fluorescent reporter.
We intended to include a marker molecule in our cassette in order to be able to track where our protein is, whether it has been transported to the membrane, and to calculate the quantification of protein.
Fluorescent labeling is the process of binding fluorescent dyes to functional groups contained in biomolecules so that they can be visualized by fluorescence imaging. GFP is one of the most commonly used targeting molecules, therefore we initially considered using it. However, because GFP is a dimeric protein and would cause stereochemical inhibition with the synthetic spike-1 protein, we chose to use a fluorescent protein with a monomeric structure. After research, we concluded that we needed to use mCherry since it has many advantages over others. While conducting our bibliographical research we came across 2019’s iGEM Team: IISc-Bangalore wiki page. The information provided there about the mCherry protein was extremely helpful and we’d wish to thank them a lot. Specifically we concluded that some of the most paramount benefits were the facts that since mCherry was created to address the inadequacies of the original DsRed fluorophore, it has a number of benefits over it.
MCherry qualifies as a good fluorophore for a variety of applications since it has one of the shortest maturation times of all known fluorophores and a relatively high brightness on adequate excitation (Extinction coefficient = 72,000 M-1 cm-1 and fluorescence quantum yield = 0.22). It is also very photostable, with a lifespan of about 1.4 nanoseconds, making storage and use of the protein considerably easier. Moreover, TexasRed and other fluorescence standards are widely available, and their spectrum overlaps with that of mCherry. TexasRed has also been offered as a chemical reference for mCherry by the iGEM measurement committee. Lastly, along with the fact that GFP is dimeric within the cell and this dimerization can create stereochemical interference in how the trimer of the spike protein will form whereas the mCherry protein doesn’t exhibit such a behaviour we chose to use mCherry as the fluorescent protein rather than GFP. We also went ahead, due to the fact that we used this fluorescent dye, and registered this protein in the registry of standard biological parts under the code: BBa K3935001.
Module 4: Transformation
In our experimental procedure we’re executing 2 bacterial transformations, one using E.coli strain DH10B and another one using Agrobacterium tumefaciens. We got our chimeric protein delivered in two parts, and ,thus, in two plasmids, by the Eurofins Genomics and we transferred it in a pBluescript II SK(-) plasmid.
The pBluescript II phagemids (plasmids with a phage origin) are cloning vectors designed to simplify commonly used cloning and sequencing procedures. The pBluescript II phagemids (plasmids with a phage beginning) are cloning vectors intended to improve on generally utilized cloning and sequencing methodology. The pBluescript II phagemids have an extensive polylinker with 21 unique restriction enzyme recognition sites.The choice of promoter used to initiate transcription determines which strand of the insert cloned into the polylinker will be transcribed.
The minus (-) in our plasmid has to do with the orientation of the f1 origin.
Our recombinant pBluescript II plasmid was inserted in E.coli bacteria. DH10B E.Coli strain was created with large-insert DNA library clones in mind. This strain is suitable for cloning DNA containing methylcytosine, 5-hydroxymethylcytosine, and methyladenine from both prokaryotic and eukaryotic cells. It has a wide range of applications due to its multiple characteristics, including excellent DNA transformation efficiency and the preservation of big plasmids and BACs. It has a high transformation efficiency DNA and enables blue/white colony management at the same time. Due to the loss of leuLABCD, DH10B requires leucine for growth in minimum medium and also has both the relA1 and spoT1 alleles, making it more sensitive to nutritional changes and having slightly lower growth rates than the wild type.
We used reporter genes to allow us to determine which of the E.coli cultures we cultivated in the lab received the plasmid and insert successfully. To detect which plasmids carry the DNA insert and which do not, we employed the white-blue option (Alpha complementation). These plasmids contain an additional gene called lac Z that encodes a portion of the enzyme β-galactosidase. When the plasmid is transformed into a suitable host that contains the gene for the rest of the enzyme, the whole enzyme can be produced and these bacteria form blue colonies in the presence of X-gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside) and an inducer of IPTG (Isopropyl β-D-Thiogalactopyronoside). Within the lac Z gene in plasmids, there are several cloning sites, and any insertion of foreign DNA into this region results in the loss of the capacity to generate active -galactosidase. Thus, colonies carrying the plasmid with the insert will remain white, while colonies carrying the plasmid without the insert will remain blue.
The Transformed E.coli cells that expressed the LacZ and Amp genes and were thus able to survive were isolated and cultivated in the appropriate medium. The isolation of our combined chimeric part from the transformed E. coli cells using gel electrophoresis is described in the experiments section.
Utilizing Agrobacterium tumefaciens
To produce edible vaccines, the gene encoding the active antigenic protein Spike 1 must first be incorporated into a suitable "gene vector". Agrobacterium tumefaciens is a naturally occurring gram-negative bacterium that is widely used as a vector in plant biotechnology to infect and incorporate the desired foreign gene into the plant tissue cell. This plasmid incorporates a portion of its DNA, known as T-DNA, into the chromosomal DNA of its host plant cells, which can be transcribed and expressed.The T-DNA consists of tumor-inducing genes in the infected cells and genes associated with its transportation into the plant cell.
However, in genetic manipulation, the genes that cause tumour growth in plants are removed from the Ti plasmid, making the plasmid safe. Thus, the genes in the Ti plasmid responsible for the production of proteins that contribute to T-DNA transport are retained while the unwanted genes are turned off.
At the same time, the gene encoding the desired protein antigen for the vaccine is incorporated for practical purposes into an alternative plasmid called pICSL86922. This plasmid provides the selection markers necessary for the selection of bacteria containing the desired gene in culture and the genes necessary for its expression in eukaryotic cells.
However, the alternative plasmid cannot be transferred by itself into the nucleus of the plant cell but requires the presence of the Ti plasmid; that is, as Ti is transferred, so is pICSL86922, which carries the gene encoding the protein antigen.
Thus, this gene vehicle is incorporated into the genome of the plant and allowed to express the corresponding antigen. Finally, these parts of the plants feed on animals and humans causing immunity against SARS-CoV-2.
Transformation of Agrobacterium tumefaciens
We wanted to transform A.tumefaciens bacteria with the pICH86988 plasmid, into which we inserted -among other genes- the cassette that is responsible for the expression of our antigen. For the transformation procedure we followed the ‘heat shock’ technique, which was analysed previously. In our plasmid -pICH86988- we inserted a number of genes such as the cassette responsible for the expression of the trimerized spike 1 protein, genes that refer to the transfer of the cassette from the bacterium to the plant cell, and selection markers. Plasmids carry one or more selection markers - genes that confer antibiotic resistance- , so if the transformation is successful, which means that the plasmid is inserted and copied into the host, the host cell will successfully develop in the presence of antibiotics. Selection markers are often used in ampicillin (amp), tetracycline (tet), kanamycin (kan), streptomycin and chloramphenicol. The cells are plated in a selective growth media, typically petri plates with agar containing nutritional medium and the antibiotic to which the plasmid confers resistance. In our case, the plasmid has the ampicillin resistance gene, so the petri dish will contain ampicillin, and bacteria that have been successfully transformed and carry the plasmid will survive and reproduce in ampicillin-containing plates. The plasmid will be replicated in bacteria and passed down across generations. The chosen cultures will then be injected into lettuce leaf cells using the agroinfiltration method.
Module 5: Agroinfiltration Technique
Confocal microscopy is a technique that creates a three-dimensional image of a sample using lasers and fluorescence. To excite the molecules at one spot in the sample, a concentrated laser beam is used. The fluorescence is caused by photons released by the fluorophores as they return to their unexcited condition. An image of the sample can be formed by scanning it across it. We utilized this strategy to assess mCherry protein. You can see the results by clicking on this link.
Module 6: Agroinfiltration Technique
Purpose, Mechanism, Benefits
Following the insert of our cassette into the vector through the transformation process, the next step was to choose an infusing technique of our bacteria in the lettuce leaves. Although variability in such methods, after collecting data from a bunch of scientific papers we came to the conclusion that the most suitable method for this purpose is the method of Agroinfiltration, as it has many benefits.
Agrobacterium Excretory System
Has a type IV explosive mechanism that can transfer DNA into the nucleus of the host cell and start expressing the gene within. The expression of the chimeric protein occurs once the agrobacterium is amplified in plant tissue, and we monitor its production using protein fluorescence. The tissue around the agroinfiltration spot where the desired protein was generated was then cut. The benefit in this scenario is that we avoid using genetically modified plants (GMOs), which reduces the ethical difficulties we would otherwise face because we are not producing a genetically changed plant, but merely a portion of its plant tissue that expresses the protein.
To become a mature T-complex, the T-clone-VirD2 complex, dubbed "immature T-complex," is covered with many VirE2 molecules. The T-clone is subsequently carried into the host cytoplasm as an immature or mature T-clone, passes through the host cytoplasm, and finally enters the host nucleus via a vir B/virD4-encoded channel. VirE3 proteins are also exported to the cytoplasm of the host cell via the same VirB/VirD4 channel to aid in the nuclear import of the mature T-complex later on.
Intermediate transformation with A. tumefaciens is now a commonly utilized technique because it offers a number of benefits, including the insertion of well-defined DNA pieces, a high rate of plant transformation, and a cheap relative cost. Several successful attempts to express antigen in transitional plants by Agrobacterium-mediated nuclear transformation have been made to date; examples include the cholera toxin B subunit protein, the Norwalk virus capsid protein, and others.
Module 7: Applications in vertebrates
Premise, Problems, Solutions
Our project essentially in its simplest form aims to demonstrate the proper function of an edible vaccine. Given that our goal is to achieve transmembrane expression of our own synthetically engineered protein: Sars-Cov-2 spike S1 by transient expression in lettuce leaves through the agroinfiltration process so as to induce an immune response in humans and other vertebrates through the gut mucosa. From the beginning, we were aware of the necessity of using laboratory animals in order to prove that our vaccine actually induces an immune response in vertebrates and in particular in mice. Unfortunately, the adverse conditions of the pandemic affected us considerably, as the time remaining to carry out both experimental procedures after the end of the intensive quarantine was limited. Thus, although we had to abandon the mouse immunization experiment, we decided, with the knowledge gained from the relevant literature and from our contacts with competent professors and veterinarians, to present a theoretical expansion of this experimental procedure. Read More
Methods for administration control: Encapsulation
One of our major concerns during the design of the edible vaccine was the method of delivery. As the immunization is to take part in the intestines, the delivery of the antigens in their native form is key to the effectiveness of the procedure. This means that protection of the antigen in the acidic conditions of the stomach accounts for the first goal while the second one is the release of the antigens in the intestines in a preferably controlled manner. After conducting research, we came to the conclusion that a good method of delivery of the edible vaccine that meets the above purposes could be the use of a capsule.
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