iGEM 2021 — Team Crete

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Crete/Notebook

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

* [Home (current)](https://2021.igem.org/Team%3ACrete)
* Team

  [Members](https://2021.igem.org/Team%3ACrete/Team)
  [Attributions](https://2021.igem.org/Team%3ACrete/Attributions)
* Project

  [Description](https://2021.igem.org/Team%3ACrete/Description)
   [Contribution](https://2021.igem.org/Team%3ACrete/Contribution)
   [Engineering](https://2021.igem.org/Team%3ACrete/Engineering)
   [Implementation](https://2021.igem.org/Team%3ACrete/Implementation)
   [Proof of Concept](https://2021.igem.org/Team%3ACrete/Proof_Of_Concept)
  [Model](https://2021.igem.org/Team%3ACrete/Model)
   [Safety](https://2021.igem.org/Team%3ACrete/Safety)
  [Sustainability](https://2021.igem.org/Team%3ACrete/Sustainable)
* Lab work

  [Experiments](https://2021.igem.org/Team%3ACrete/Experiments)
   [Notebooks](https://2021.igem.org/Team%3ACrete/Notebook)
   [Results](https://2021.igem.org/Team%3ACrete/Results)
   [Parts](https://2021.igem.org/Team%3ACrete/Parts)
* [HP](https://2021.igem.org/Team%3ACrete/Human_Practices)

  [Human Practices](https://2021.igem.org/Team%3ACrete/Human_Practices)
   [Education](https://2021.igem.org/Team%3ACrete/Education)
   [Collaborations](https://2021.igem.org/Team%3ACrete/Collaborations)
   [Communication](https://2021.igem.org/Team%3ACrete/Communication)
   [Paving the future](https://2021.igem.org/Team%3ACrete/Excellence)
* Awards

  [Medals](https://2021.igem.org/Team%3ACrete/Medals)
   [Judging form](https://igem.org/2021_Judging_Form?id=3935)

⇧

Shortcut Menu

* [Lab Notebook](#Lab)

# Lab Notebook

##### **WEEK 1 (19-25/07)** * Receiving of 3 plasmids containing the 3 s1 spike sequences and 1 plasmid containing mCherry and the transmembrane domain. * Lab sorting * Lab safety training **WEEK 2 (26/07-1/08)** * Golden Gate Cloning * Start of an E.coli DH10B culture **WEEK 3 (2-8/08)** * Transformation of E.coli DH10B * Selection of bacteria transformed with the recombined vector * Culture of selected bacteria **WEEK 4 (9-15/08)** * Lab Closed **WEEK 5 (16-22/08)** * Lab closed **WEEK 6 (23-29/08)** * Isolation of plasmid DNA from recombined E.coili cultures * Amplification via PCR **WEEK 7 (30/08-5/09)** * Issue at transferring plasmid DNA from pBlueScript to pICSL86922 **WEEK 8 (6-12/09)** * Transformation of Agrobacteria Tumefaciens with plCSL86922 * Culture of Agrobacteria * Gel electrophoresis of the plasmid **WEEK 9 (13-19/09)** * Selection of transformed Agrobacteria cultures * Culture of selected bacteria **WEEK 10 (20-26/09)** * Agroinfiltration of plant leaves with transformed Agrobacteria * Confocal microscopy of plant leaves **WEEK 11 (27/09-3/10)** * Repeat of the confocal microscopy session * Further Agroinfiltration of plants to create a sufficient quantity of the edible vaccine

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

![](https://static.igem.org/mediawiki/2021/b/b3/T--Crete--UoC.png)

![](https://static.igem.org/mediawiki/2021/e/e4/T--Crete--Forth.jpeg)

## *Find us*

[![](https://static.igem.org/mediawiki/2021/c/c6/T--Crete--fb.png)](#!)
[![](https://static.igem.org/mediawiki/2021/5/53/T--Crete--insta.png)](#!)
[![](https://static.igem.org/mediawiki/2021/7/77/T--Crete--yt.png)](#!)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Crete/Engineering

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

* [Home (current)](https://2021.igem.org/Team%3ACrete)
* Team

  [Members](https://2021.igem.org/Team%3ACrete/Team)
  [Attributions](https://2021.igem.org/Team%3ACrete/Attributions)
* Project

  [Description](https://2021.igem.org/Team%3ACrete/Description)
   [Contribution](https://2021.igem.org/Team%3ACrete/Contribution)
   [Engineering](https://2021.igem.org/Team%3ACrete/Engineering)
   [Implementation](https://2021.igem.org/Team%3ACrete/Implementation)
   [Proof of Concept](https://2021.igem.org/Team%3ACrete/Proof_Of_Concept)
  [Model](https://2021.igem.org/Team%3ACrete/Model)
   [Safety](https://2021.igem.org/Team%3ACrete/Safety)
  [Sustainability](https://2021.igem.org/Team%3ACrete/Sustainable)
* Lab work

  [Experiments](https://2021.igem.org/Team%3ACrete/Experiments)
   [Notebooks](https://2021.igem.org/Team%3ACrete/Notebook)
   [Results](https://2021.igem.org/Team%3ACrete/Results)
   [Parts](https://2021.igem.org/Team%3ACrete/Parts)
* [HP](https://2021.igem.org/Team%3ACrete/Human_Practices)

  [Human Practices](https://2021.igem.org/Team%3ACrete/Human_Practices)
   [Education](https://2021.igem.org/Team%3ACrete/Education)
   [Collaborations](https://2021.igem.org/Team%3ACrete/Collaborations)
   [Communication](https://2021.igem.org/Team%3ACrete/Communication)
   [Paving the future](https://2021.igem.org/Team%3ACrete/Excellence)
* Awards

  [Medals](https://2021.igem.org/Team%3ACrete/Medals)
   [Judging form](https://igem.org/2021_Judging_Form?id=3935)

⇧

Shortcut Menu

* [Engineering success for the development of an edible vaccine](#success)
* [Module 1: Theoretical experiment and design](#Mod1)
* [Module 2: Golden Gate Cloning](#Mod2)
* [Module 3: Chimeric protein expression](#Mod3)
* [Module 4: Transformation](#Mod4)
* [Module 5: Confocal Imaging](#Mod5)
* [Module 6: Agroinfiltration Technique](#Mod6)
* [Module 7: Applications in vertebrates](#Mod7)

# 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

![](https://static.igem.org/mediawiki/2021/0/0f/T--Crete--eng9.png)

##### 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.

![](https://static.igem.org/mediawiki/2021/f/fc/T--Crete--eng1.png)

##### 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.

![](https://static.igem.org/mediawiki/2021/6/65/T--Crete--eng2.png)

##### 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.

![](https://static.igem.org/mediawiki/2021/6/6a/T--Crete--eng3.png)

##### **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. ![](https://static.igem.org/mediawiki/2021/1/19/T--Crete--eng4.png) **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.

[Read More](#multiCollapseExample1)

![](https://static.igem.org/mediawiki/2021/6/68/T--Crete--eng5.png)

# 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](2021.igem.org/Team%3ACrete/Model).

### 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](2021.igem.org/Team%3ACrete/Human_Practices) 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.

![](https://static.igem.org/mediawiki/2021/5/59/T--Crete--eng6.png)

![](https://static.igem.org/mediawiki/2021/2/25/T--Crete--eng7.png)

[Read More](#multiCollapseExample2)

![](https://static.igem.org/mediawiki/2021/4/43/T--Crete--eng8.png)

### 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.

![](https://static.igem.org/mediawiki/2021/4/49/T--Crete--eng10.png)

##### 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**.

[Read More](#multiCollapseExample3)

![](https://static.igem.org/mediawiki/2021/f/f7/T--Crete--eng11.png)

# 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.

![](https://static.igem.org/mediawiki/2021/4/4b/T--Crete--eng41.png)

##### 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.

![](https://static.igem.org/mediawiki/2021/6/69/T--Crete--eng42.png)

##### 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](https://2021.igem.org/Team%3ACrete/Experiments).

[Read More](#multiCollapseExample55)

![](https://static.igem.org/mediawiki/2021/f/f9/T--Crete--eng43.png)

### 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.

![](https://static.igem.org/mediawiki/2021/6/66/T--Crete--eng44.png)

##### 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.

![](https://static.igem.org/mediawiki/2021/a/a2/T--Crete--eng45.png)

##### 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.

[Read More](#multiCollapseExample51)

![](https://static.igem.org/mediawiki/2021/0/06/T--Crete--eng46.png)

### 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

![](https://static.igem.org/mediawiki/2021/9/9d/T--Crete--res5.gif)

##### 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](https://2021.igem.org/Team%3ACrete/Results).

# 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.

[Read More](#multiCollapseExample4)

![](https://static.igem.org/mediawiki/2021/4/47/T--Crete--eng52.png)

### 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.

![](https://static.igem.org/mediawiki/2021/0/08/T--Crete--eng53.png)

##### 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.

[Read More](#multiCollapseExample31)

![](https://static.igem.org/mediawiki/2021/4/4f/T--Crete--eng54.png)

### Conclusion

##### 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](https://2021.igem.org/Team%3ACrete/Proof_of_Concept)

### 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.

[Read More](#multiCollapseExample47)

![](https://static.igem.org/mediawiki/2021/c/ca/T--Crete--eng00.png)

### References

##### * Lourdes Gómez-Gómez, Thomas Boller,FLS2: An LRR Receptor-like Kinase Involved in the Perception of the Bacterial Elicitor Flagellin in Arabidopsis, Molecular Cell, Volume 5, Issue 6, 2000, Pages 1003-1011, ISSN 1097-2765, https://doi.org/10.1016/S1097-2765(00)80265-8. * Sun Y, Li L, Macho AP, Han Z, Hu Z, Zipfel C, Zhou JM, Chai J. Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science. 2013 Nov 1;342(6158):624-8. doi: 10.1126/science.1243825. Epub 2013 Oct 10. PMID: 24114786. * World Health Organization (WHO). WHO Coronavirus (COVID-19) Dashboard. WHO Coronavirus Disease (COVID-19). Available online: https://covid19.who.int/table * Capell, T.; Twyman, R.M.; Armario-Najera, V.; Ma, J.K.-C.; Schillberg, S.; Christou, P. Potential Applications of Plant Biotechnology against SARS-CoV-2. Trends Plant Sci. 2020, 25, 635–643. * Bibliography: Biolabs, New England. “Golden Gate Assembly.” New England Biolabs: Reagents for the Life Sciences Industry, * Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. PLoS ONE 4, e5553 (2009). * https://www.youtube.com/watch?v=xusiavAC\_Xk&ab\_channel=synbio%40tsl * https://2012.igem.org/Team:Freiburg/Project/Golden * Chen Q, Dent M, Hurtado J, Stahnke J, McNulty A, Leuzinger K, Lai H. Transient Protein Expression by Agroinfiltration in Lettuce. Methods Mol Biol. 2016;1385:55-67. doi: 10.1007/978-1-4939-3289-4\_4. PMID: 26614281. * Joh LD, Wroblewski T, Ewing NN, VanderGheynst JS. High-level transient expression of recombinant protein in lettuce. Biotechnol Bioeng. 2005 Sep 30;91(7):861-71. doi: 10.1002/bit.20557. PMID: 15937952. * Wesołowska A, Kozak Ljunggren M, Jedlina L, et al. A Preliminary Study of a Lettuce-Based Edible Vaccine Expressing the Cysteine Proteinase of Fasciola hepatica for Fasciolosis Control in Livestock. Front Immunol. 2018;9:2592. Published 2018 Nov 13. doi:10.3389/fimmu.2018.02592 * Kurup VM, Thomas J. Edible Vaccines: Promises and Challenges. Mol Biotechnol. 2020;62(2):79-90. doi:10.1007/s12033-019-00222-1 * Criscuolo E, Caputo V, Diotti RA, Sautto GA, Kirchenbaum GA, Clementi N. Alternative Methods of Vaccine Delivery: An Overview of Edible and Intradermal Vaccines. J Immunol Res. 2019;2019:8303648. Published 2019 Mar 4. doi:10.1155/2019/8303648 * Saxena J, Rawat S. Edible Vaccines. Advances in Biotechnology. 2013;207-226. Published 2013 Oct 22. doi:10.1007/978-81-322-1554-7\_12 * Tzfira T, Lacroix B, Citovsky V. Nuclear Import of Agrobacterium T-DNA. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6493/?fbclid=IwAR1zi1Na8k1K-iBaMlXw99CzJt1f8kdVae7oRnvS5M2vu\_Fch-z9CJxvlNU * Subach, F., Patterson, G., Manley, S. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6, 153–159 (2009). https://doi.org/10.1038/nmeth.1298 * Bin Wu, Yan Chen, Joachim D. Müller, Fluorescence Fluctuation Spectroscopy of mCherry in Living ISSN 0006-3495, https://doi.org/10.1016/j.bpj.2008.12.3902.

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

![](https://static.igem.org/mediawiki/2021/b/b3/T--Crete--UoC.png)

![](https://static.igem.org/mediawiki/2021/e/e4/T--Crete--Forth.jpeg)

## *Find us*

[![](https://static.igem.org/mediawiki/2021/c/c6/T--Crete--fb.png)](#!)
[![](https://static.igem.org/mediawiki/2021/5/53/T--Crete--insta.png)](#!)
[![](https://static.igem.org/mediawiki/2021/7/77/T--Crete--yt.png)](#!)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Crete/Experiments

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

* [Home (current)](https://2021.igem.org/Team%3ACrete)
* Team

  [Members](https://2021.igem.org/Team%3ACrete/Team)
  [Attributions](https://2021.igem.org/Team%3ACrete/Attributions)
* Project

  [Description](https://2021.igem.org/Team%3ACrete/Description)
   [Contribution](https://2021.igem.org/Team%3ACrete/Contribution)
   [Engineering](https://2021.igem.org/Team%3ACrete/Engineering)
   [Implementation](https://2021.igem.org/Team%3ACrete/Implementation)
   [Proof of Concept](https://2021.igem.org/Team%3ACrete/Proof_Of_Concept)
  [Model](https://2021.igem.org/Team%3ACrete/Model)
   [Safety](https://2021.igem.org/Team%3ACrete/Safety)
  [Sustainability](https://2021.igem.org/Team%3ACrete/Sustainable)
* Lab work

  [Experiments](https://2021.igem.org/Team%3ACrete/Experiments)
   [Notebooks](https://2021.igem.org/Team%3ACrete/Notebook)
   [Results](https://2021.igem.org/Team%3ACrete/Results)
   [Parts](https://2021.igem.org/Team%3ACrete/Parts)
* [HP](https://2021.igem.org/Team%3ACrete/Human_Practices)

  [Human Practices](https://2021.igem.org/Team%3ACrete/Human_Practices)
   [Education](https://2021.igem.org/Team%3ACrete/Education)
   [Collaborations](https://2021.igem.org/Team%3ACrete/Collaborations)
   [Communication](https://2021.igem.org/Team%3ACrete/Communication)
   [Paving the future](https://2021.igem.org/Team%3ACrete/Excellence)
* Awards

  [Medals](https://2021.igem.org/Team%3ACrete/Medals)
   [Judging form](https://igem.org/2021_Judging_Form?id=3935)

⇧

Shortcut Menu

* [Preparation of Primers](#Primers)
* [Preparation of dried DNA](#DNA)
* [Starting a liquid culture of bacteria](#Bacteria)
* [Transformation of competent cells](#Cells)
* [Isolation of plasmid DNA](#Isolation)
* [PCR with Phusion® HighFidelity- DNA Polymerase](#Polymerase)
* [Gel extraction and purification of PCR products](#Gel)
* [Adding T-ends](#T-ends)
* [T4 Ligation](#Ligation)
* [Transformation of Agrobacterium Tumefaciens](#Tumefaciens)
* [Agroinfiltration](#Agroinfiltration)

# Preparation of Primers

##### When the primers arrive, we give them a spin to settle any residue on the lid. We then dissolve the powder very well in water that has been recently sterilized and opened at that time. In addition, we also use new tips. We follow the instructions given in the leaflet that comes with the primers so that the final concentration is 100 μM. From the stock solutions we make aliquots with a concentration of 10 μM. We keep them at -20°C.

# Preparation of dried DNA

##### If the DNA is dry we do a spin. Then we redissolve it well under a flame. To assist the redissolution we place it in a water bath at 30°C for 5 min. Afterwards, we keep it on ice. To store it, put it in the refrigerator.

# Starting a liquid culture of bacteria

##### The various bacterial strains as well as bacteria carrying specific plasmids are stored in glycerol stocks at -80°C. - You will need a gas, lighter, LB (lysogenic broth), falcon tubes, ampicillin (in a waterbath to defrost faster), and tips which you only use under a flame (note this on the box). - First, we prepare in a falcon tube the entire amount of LB we will need by adding ampicillin so that its final concentration in the solution is 100 μg/ml. - Add the appropriate amount of LB to the falcon tubes. Always make sure that there is enough ventilation in the tube. For this purpose, the volume remaining free in the tube should be at least a little more than twice the volume of LB added (e.g. in a 15-well facon we put up to 4 ml LB). - We take ice and put in the appropriate glycerol stocks. In case we want more than two different samples, we take them out of the freezer half and half so they don't stay out for a long time. - We work under a flame. - The tips should be located to the right of the gas and opened so that they face the flame. - Open the falcon tube and place the lid on the gas under the flame. - Open the eppendorf carefully to avoid droplets. - Put tip in a pipette (200 or 20), put it in the eppendorf tube, shake it around to get a small amount, and pour it all into the LB in the falcon tube. - Close the lid after passing it over the flame. - Place the falcon tubes in a beaker and add additional crinkled paper, making sure they do not shake. Cover the beaker with foil and tape around it with cellophane. - E. coli are incubated overnight at 200 rpm and 37°C, while Agrobacterium tumefaciens are incubated at 200 rpm at 28°C.

### Golden Gate Cloning

##### For the assembly you will need quantified aliquots of DNA containing each assembly piece, as well as the vector. If assembly pieces are cloned in level 0 vectors (plasmids) use 100ng of the vector backbone and EQUIMOLAR\* amounts of the other assembly pieces to a 15μl total volume assembly reaction mixture

![](https://static.igem.org/mediawiki/2021/f/fa/T--Crete--exp1.png)

##### Backbone vector: pBluescript\II\SK(-)\GG [[BBa\_K3935000]](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3935000) Use 10μl for transformation of E. coli competent cells and plate for Ampicillin selection. Also, selection can be made using xGal as this vector contains a lacZ gene.

# Transformation of competent cells

##### A total of 10 μL of the ligation reaction was transformed into chemically competent DH10B cells. After incubation at 4°C for 30 min, the cells were heat-shocked at 42°C for 1 min and returned immediately to 4°C for 1 min. Then, LB was added up to a total volume of 1 mL. After incubation at 37°C with shaking at 200 rpm for 1 h, the cells were centrifuged at 2100 × g for 2 min, the supernatant was discarded, and the pellet was resuspended in the remaining solution, plated on solid LB with ampicillin (100 μg/mL) and incubated at 37°C overnight.

# Isolation of plasmid DNA

##### **Alkaline Lysis Buffer I:** Glucose 50 mM Tris-Cl (pH 8.0) 25 mM EDTA (pH 8.0) 10 mM It is prepared whenever the previous one is finished and kept in the refrigerator. **Alkaline Lysis Buffer II:** NaOH 0.2 N SDS 1% w/v Make it every time, unless it is reused within a month, and keep it at room temperature. **Alkaline Lysis Buffer III:** Potassium acetate 5 M: 60 ml Glacial acetic acid: 11.5 ml H2O: 28.5 ml It is kept in the refrigerator. We will need a rack of tips to pour the liquid waste, a rack for the tubes, eppendorf tubes, the solutions listed above, ice, 70% EtOH, and 100% EtOH. Cultures of E. coli in LB were incubated overnight at 37°C, 200 rpm until saturation (concentration more than 109 cells/mL). The saturated cultures were centrifuged at 11000 × g, 25°C, for 30 s. The supernatant was discarded, and the pellet was resuspended by vortexing in 100 μL alkaline lysis solution I, pH 8.0 (glucose 50 mM, Tris-HCl 25 mM, EDTA 10 mM, 4°C) per 3 mL of culture. Then, 200 μL of alkaline lysis solution II (NaOH 0.2 N, SDS 1% w/v, 25°C) and 150 μL of alkaline lysis solution III (60 mL potassium acetate 5 M, 11.5 mL glacial acetic acid, 28.5 mL H2O, 4°C) were added per 3 mL of culture. Each sample was mildly mixed after the addition of solutions II and III. After incubation at 4°C for 3 min, it was centrifuged at 13100 × g for 5 min at 4°C. The supernatant was transferred to a clean tube, and the DNA was precipitated with two volumes of absolute ethanol by shaking, incubating at 25°C for 2 min, and centrifuging at 13100 × g for 15 min. The supernatant was discarded, and the pellet washed with the addition of 500 μL 70% v/v ethanol and centrifuged at 13100 × g for 5 min. Finally, the ethanol was removed, and the pellet was dried and resuspended in 30 μL sterile distilled water (dH2O).

# PCR with Phusion® HighFidelity- DNA Polymerase

##### Template DNA 1 ng: **1 μl** 5X Phusion Buffer: **10 μl** Fw Primer (0.5 μM): **2.5 μl** Rv Primer (0.5 μM): **2.5 μl** dNTPs (0.2 mM): **1 μl** Phusion DNA Polymerase (2 U/μl): **0.5 μl** H2O: **32.5 μl** Total: **50 μl**

![](https://static.igem.org/mediawiki/2021/6/65/T--Crete--exp2.png)

# Gel extraction and purification of PCR products

##### - Turn on the water bath at 48°C. - We use a different scalpel for each sample and transfer each piece of gel to a different eppendorf tube. The tube is grasped without a glove to avoid transferring ethidium. - For every 100 mg of ≤ 2% w/v agarose gel, 200 μl of NTI Buffer is added and the samples are incubated at 48°C until the gel is completely melted. At intervals, assist the dissolution with vortex. We then follow the NucleoSpin® Gel and PCR Clean-up protocol. - The final elution is performed with 30 μl H2O.

# Adding T-ends

##### Template DNA (all the amount isolated): **30 µl** 10X Taq DNA Polymerase Buffer: **5 μl** dATPs (0.5 mM): **2.5 μl** Taq DNA Polymerase (5 U/μl): **0.2 μl** ddH2O: **12.3 μl** Total: **50 μl** - Incubate at 72°C for 30 min (PCR machine). - Quantify by loading 1 μl of DNA, 1 μl of Orange G, and 4 μl of H2O onto the gel mixture and running it at 100 V for 15 min.

# T4 Ligation

##### The amount of DNA insert needed is calculated from the following formula:

![](https://static.igem.org/mediawiki/2021/f/f9/T--Crete--exp3.png)

##### The amount of vector used is 50 ng and the sizes of the vector and insert DNA in bp are also known. Usually the vector/insert ratio is 3 molecules of insert for one molecule of vector (3:1). Thus, the only unknown quantity is the amount of insert DNA. 10X T4 DNA Ligase Buffer: **2 μl** vector DNA (50 ng): **1 μl (pICSL86922)** insert DNA (minsert): **59.4 ng** H2O: \_\_ (so that the final volume is 20 µl) T4 DNA Ligase: **1 μl** Total: **20 μl** - Incubate at room temperature for 2 h.

# Transformation of Agrobacterium Tumefaciens

##### Preparation of Competent Cells

##### 1. Inoculate 200 ml of LB media with 1 ml of an overnight culture of the chosen strain of Agrobacterium. Incubate at 28ºC with vigorous agitation.The culture was created in the late afternoon and harvested the next morning 2. The cells were grown to log phase (OD 550 0.5-0.8). 3. Pellet the cells in a benchtop centrifuge at 5000 x g for 10 minutes at room temperature. 4. Wash the pellet with sterile 1X TE. 5. Resuspend the cells in 0.1X the original volume of LB, and aliquot 250μl fractions in microcentrifuge tubes. 6. Snap-freeze in liquid nitrogen and store at -70ºC.

##### Transformation

##### 7. Thaw competent Agrobacterium on ice (use 250 μl per transformation reaction), and add DNA (up to 10 μl, 100-1000ng) and flick tube gently to mix. 8. Keep the mixture on ice for 5 minutes, and then transfer to liquid nitrogen for 5 minutes. 9. Incubate the mixture for an additional 5 minutes in a 37ºC water bath. 10. Transfer 250 μl of cells to a 15 ml Corning tube containing 1 ml of LB and shake at 28ºC for 2 hours. 11. Collect the cells by spinning 2 min at 5000 rpm, and resuspend cells in 100-200 µl LB. Spread them on two LB agar plates containing the ampicillin(100 μg/mL). 12. Incubate the cells for 2 days at 26-28ºC for colonies to form.

# Agroinfiltration

##### The solution is placed in a needleless syringe for injection(,without a needle). The syringe tip is placed against the underside of a leaf while mild counter pressure is applied to the opposite side of the leaf. The Agrobacterium suspension is then injected into the leaf's airspaces via stomata, or sometimes a small incision on the underside of the leaf.

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

![](https://static.igem.org/mediawiki/2021/b/b3/T--Crete--UoC.png)

![](https://static.igem.org/mediawiki/2021/e/e4/T--Crete--Forth.jpeg)

## *Find us*

[![](https://static.igem.org/mediawiki/2021/c/c6/T--Crete--fb.png)](#!)
[![](https://static.igem.org/mediawiki/2021/5/53/T--Crete--insta.png)](#!)
[![](https://static.igem.org/mediawiki/2021/7/77/T--Crete--yt.png)](#!)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Crete/Results

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

* [Home (current)](https://2021.igem.org/Team%3ACrete)
* Team

  [Members](https://2021.igem.org/Team%3ACrete/Team)
  [Attributions](https://2021.igem.org/Team%3ACrete/Attributions)
* Project

  [Description](https://2021.igem.org/Team%3ACrete/Description)
   [Contribution](https://2021.igem.org/Team%3ACrete/Contribution)
   [Engineering](https://2021.igem.org/Team%3ACrete/Engineering)
   [Implementation](https://2021.igem.org/Team%3ACrete/Implementation)
   [Proof of Concept](https://2021.igem.org/Team%3ACrete/Proof_Of_Concept)
  [Model](https://2021.igem.org/Team%3ACrete/Model)
   [Safety](https://2021.igem.org/Team%3ACrete/Safety)
  [Sustainability](https://2021.igem.org/Team%3ACrete/Sustainable)
* Lab work

  [Experiments](https://2021.igem.org/Team%3ACrete/Experiments)
   [Notebooks](https://2021.igem.org/Team%3ACrete/Notebook)
   [Results](https://2021.igem.org/Team%3ACrete/Results)
   [Parts](https://2021.igem.org/Team%3ACrete/Parts)
* [HP](https://2021.igem.org/Team%3ACrete/Human_Practices)

  [Human Practices](https://2021.igem.org/Team%3ACrete/Human_Practices)
   [Education](https://2021.igem.org/Team%3ACrete/Education)
   [Collaborations](https://2021.igem.org/Team%3ACrete/Collaborations)
   [Communication](https://2021.igem.org/Team%3ACrete/Communication)
   [Paving the future](https://2021.igem.org/Team%3ACrete/Excellence)
* Awards

  [Medals](https://2021.igem.org/Team%3ACrete/Medals)
   [Judging form](https://igem.org/2021_Judging_Form?id=3935)

⇧

Shortcut Menu

* [Results](#Results)
* [Gel](#Gel electrophoresis)
* [Confocal Microscopy](#Confocal)

# Results

##### 2021 was a challenging year for all iGEM Teams. Despite the fact that our country was under lockdown for half a year, we were lucky enough to enter the laboratory in the middle of summer, conduct most of our experiments and get some promising results! Our wet lab team brings two deliverables for the 2021 iGEM competition. The first being DNA gel electrophoresis and the second being confocal microscopy.

# Gel electrophoresis

##### We performed the gel electrophoresis so as to find out whether our final plasmid vector has assimilated the insert of interest. We used BenchLing to virtually ligate with BamHI and XhoI our final plasmid vector and predict the length of the DNA fragments. The expected fragments are 6462bp, 2760bp, 1710bp and 914bp.

![](https://static.igem.org/mediawiki/2021/8/87/T--Crete--res1.png)

##### The results of our electrophoresis are shown below:

![](https://static.igem.org/mediawiki/2021/0/0a/T--Crete--res2.png)

##### In the middle there are two different DNA ladders and on the left and right side of the markers are the DNA samples. The left DNA ladder is the λ DNA/PstI and the right one is the λ DNA/StyI. As we can see in all of the DNA sample tests we have similar results, there are 4 different DNA fragments in the expected positions, so we can safely assume that the DNA fragments have the right length.

# Confocal Microscopy

##### The entirety of the project is based on the transmembrane expression of our chimeric protein in lactuca sativa leaves. This protein is fused with an mCherry fluorescent protein in its carboxy terminal part. So, in order to figure out whether the protein is expressed and placed in the anticipated manner, we carried out a confocal microscopy session after the agroinfiltration commenced and sufficient time for the protein to be expressed was given. The results are the following:

![](https://static.igem.org/mediawiki/2021/b/bb/T--Crete--res3.png)

##### In the 1st row, we can see the plant cells with the contrast being such that any fluorescent molecules can not be seen. Following that, there is the control of the confocal session, with that being the chlorophyll molecules emitting green light at 683 nm. Then we see mCherry, which is part of the protein we are interested in emitting reddish light at 610 nm. Adding the first 3 rows, we can see in the 4th row, that the protein is expressed, and is located only in the membranes of the plant , mostly in the plasma membrane with signs of expression being present in the nucleus too. A week later, we repeated the confocal microscopy session and we received the following result:

![](https://static.igem.org/mediawiki/2021/b/b4/T--Crete--res4.png)

##### In the second column we can observe the specinent depicted without the contrast, in the first with contrast and in the third being the two ones combined. We also performed a z-stack view, in which we can observe different z distances from the objective. As we can see the expression of the chimeric protein is visible in most of the z-axis distances.

![](https://static.igem.org/mediawiki/2021/9/9d/T--Crete--res5.gif)

##### From these previous observations we can confidently say that our **protein is produced in sufficient amounts**and in adequate quality.

![](https://static.igem.org/mediawiki/2021/2/26/T--Crete--log_png.png)

![](https://static.igem.org/mediawiki/2021/b/b3/T--Crete--UoC.png)

![](https://static.igem.org/mediawiki/2021/e/e4/T--Crete--Forth.jpeg)

## *Find us*

[![](https://static.igem.org/mediawiki/2021/c/c6/T--Crete--fb.png)](#!)
[![](https://static.igem.org/mediawiki/2021/5/53/T--Crete--insta.png)](#!)
[![](https://static.igem.org/mediawiki/2021/7/77/T--Crete--yt.png)](#!)