iGEM 2021 — Team UNIZAR

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# Team:UNIZAR/Notebook

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### **Notebook**

# Writing the project down

From March to October we have developed the amazing project Reconby, many weeks of hard work, long nigths and lots of fun. Here we have summarized the different activities we have done during this time and how we organized our work through these months!

|  |  |  |  |
| --- | --- | --- | --- |
| **Week 1 (March 1 to 7)**   After being chosen as members of the first UNIZAR iGEM team, we had our first online meeting and finally got to know each other in-person. | **Week 2 (March 7 to 14)**   We started working on the roster reveal for social media and some team members were officially announced on Twitter, Instagram and Facebook. | **Week 3 (March 15 to 21)**   We had our first project brainstorming meeting and initial task distribution. As well as, we recorded and edited our presentation videos. | **Week 4 (March 22 to 28)**   Proposed projects selection of final candidates. We chose four final candidates to focus our research. |
| **Week 5 (March 29 to April 4)**   *Easter Holidays.* | **Week 6 (April 5 to 11)**   Definitive project selection after a long online meeting debating pros and cons. | **Week 7 (April 12 to 18)**   We focused our research on defining more specific details of our final project. We increased our search for funding efforts contacting with some of the most important local companies and institutions related to the biological industry. | **Week 8 (April 19 to 25)**   We consulted Luis Alberto Anel professor of Biochemistry and Molecular and Cellular Biology at the University of Zaragoza, who recommended us to contact Dr. Laura Sanz, an expert in recombinant nanobodies. |
| **Week 9 (April 26 to May 2)**   We had our first "iberian meeting" with other Spanish iGEM teams. It was our first opportunity to present our projects to each other and to exchange our views on the iGEM competition and some of the problems we had encountered so far. | **Week 10 (May 3 to 9)**   We continued developing a more detailed guideline of our wet lab project as we kept looking for funding. We held our first online meeting with Operon and other companies. | **Week 11 (May 10 to 16)**   University of Zaragoza's student Vice-Rectorate office agree to pay the entire registration fee. However we kept working on finding more sponsors to cover other fees and laboratory materials and reagents. | **Week 12 (May 17 to 23)**   We kept working in our exciting project while we were looking for more sponsors. |
| **Week 13 (May 24 to 30)**   After weeks of reaseach and taking to experts in recombinant nanobodies such as Dr. Laura Sanz we got to the conclusion that our original project was unfeasible. However, we found a way to adapt our project design towards a more realistic approach, maintaining its backbone: creating a library of artificially developed nanobodies obtained by random in vitro recombination of the different CDR genetic sequences of already characterized nanobodies, and express them in *E. coli*. Moreover, we registerde our inscription and officially became part of the iGEM competition. | **Week 14 (May 31 to June 6), Week 15 (June 7 to June 13 ), Week 16 ( June 13 to 20)**   We were taking our final exams, therefore we stopped iGEM activity for two weeks. | **Week 17 (June 21 to 27)**   After our exams, we started planning our wet lab and other issues such as our education activities. In addition, our plasmids arrived at our lab. | **Week 18 (June 28 to July 4)**   Diego, Olga, Carla and Alba, responsible for web development, met up to decide some of the basic feature of our future web such as format, form or colours. In addition, Diego, Ainhoa and Alba Pueyo had a dry lab meeting to check our primers design and sequences allingments and changes. |
| **Week 19 (July 5 to 11)**   We arranged a meeting with SynBio Amazonas a group of graduated and undergraduated scientist who also worked with nanobodies and had participated in the iGEM competition. | **Week 20 (July 12 to 18)**   As a iGEM requirement, we recorded the presentation video Victor edited. | **Week 21 (July 19 to 25)**   In July 20th, we took our biosafety and biosecurity in orde to improve safety in our project. Moreover, responsibles for web development had a workshop about wiki design with the 2020 UPF team. | **Week 22 (July 26 to August 1)**   An article about our project was published at "Heraldo de Aragón", Aragon's most well-known regional newspaper. We as well created a survey to get feedback from overall population and we went out to the streets of Zaragoza to interview people who passed by. |
| **Week 23 (August 2 to 8)**   In August 7th, we installed two communication stands in two Aragonese capital cities, Teruel and Huesca. Afterwards, "Eco Teruel" published an article about us. | **Week 24 (August 9 to 15)**   During a long meeting, our team divided tasks while we were planning to start our wetlab. | **Week 25 (August 16 to 22)**   We were told that we would be able to start our lab work next week so we had a meeting to define tasks distribution and each team member assignments. | **Week 26 (August 23 to 29)**   Finally, we started our wetlab work! Materials and reagents finally arrived and we were ready to get started in the lab. We resuspended our primers and started to test materials and kits. |
| **Week 27 (August 30 to September 5)**   We placed another stand with experiments in another town in our region: Calatayud. | **Week 28 (September 6 to 12)**   Concurrently to our wetlab work, we did a survey data analysis. | **Week 29 (September 13 to 19)**   We had a meeting with Beatriz Latre to improve our science communication and put in practice what we had learnt in the next experiments stand we placed in our fourth destination: Monzón, Huesca, Zaragoza and Teruel. | **Week 30 (September 20 to 26)**   We participated in the European Researchers' Night in Zaragoza. As well, we placed a final science communication stand in our city centre. We had  [human practices](https://2021.igem.org/Team%3AUNIZAR/Human_Practices)  meeting with Dr. Esteban Martínez and Dr. Rosa Monge. |
| **Week 31 (September 27 to October 3)**   With education activities finished, we kept our focus on wetlab work. We also filled and sent the Safety Form and the preliminary Judging Form. We had [human practices](https://2021.igem.org/Team%3AUNIZAR/Human_Practices) meeting with Dr. Susana Gamen. | **Week 32 (October 4 to October 10)**   We tried to make the most of our finals days in the lab and reinforced our wiki editing. Alba Pueyo wrote the scaffold of the Team Presentation Video script. | **Week 33 (October 11 to October 17)**   Wiki text writing and checking and wiki editing. | **Week 34 (October 18 to October 24)**   Final wiki editing |

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# Team:UNIZAR/Engineering

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    [Model](https://2021.igem.org/Team%3AUNIZAR/Model)
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###### **Engineering**

# Overview

Nanobodies are proteins that **selectively** bind to an antigen. Due to their smaller size, they present advantages in diagnosis and therapeutics compared to antibodies. Their specificity relies on their **complementarity-determining regions** or CDRs (CDR1, CDR2, and CDR3), unique nanobody regions involved in antigen binding. Inspired by nature, we aim to recombine *in vitro* the different CDR genetic sequences of already characterized nanobodies at random. Our goal is to create a library of artificially developed nanobodies that bind to a specific antigen. These newly formed nanobody gene fragments are expressed in *E. coli,* generating a library for the **screening** of nanobodies with higher binding **affinity** than the starting ones. This way, our system improves the traditional method for nanobody production, being faster, cheaper, and without using animals.

**Engineering cycle**
![](https://static.igem.org/mediawiki/2021/f/f5/T--UNIZAR--Eng11.png)

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/c/c9/T--UNIZAR--Eng12.png)

## RESEARCH. How does nanobody formation happen in camelids lymphocytes?

The natural process of nanobody formation consists of recombination of **three different fragments called V, D and J** (1). There are 17 different V regions, 8 Ds and 7 Js in Alpaca's (*Vicugna pacos*) genome that can form these nanobodies (2). The recombination of these regions creates a new nanobody with **three CDRs** (Complementary Determining Regions) that allow it to recognize a certain epitope of an antigen.

Usually, synthetic nanobodies libraries are done by randomizing CDRs from a stable nanobody called scaffold (3). From our point of view, this strategy has a problem, the 3D nanobody structure doesn´t change as it depends on FR regions. We want to tackle this limitation.

![Figure 1](https://static.igem.org/mediawiki/2021/9/9a/T--UNIZAR--Eng13.png)

Figure 1. Nanobody structure. In blue, green and red are represented CDRs 1, 2 and 3 respectively.

![](https://static.igem.org/mediawiki/2021/a/aa/T--UNIZAR--Eng14.png)

## IMAGINE. How can we design a nanobodies library by mimiquing the natural process that occurs *in vivo*?

Our idea at first was to mimic what happens in nature and artificially recombine these fragments by Gibson Assembly or similar methods. We wanted to have all the possibles V, D and J regions with **homologous fragments** to perform this recombination.

![](https://static.igem.org/mediawiki/2021/3/38/T--UNIZAR--Eng3.png)

## DESIGN. What homologous regions are going to be used?

We soon found out that it would not work as **CDR3 is located in between fragments D and J**. CDR3 sequences have a really high variability as terminal deoxynucleotidyl transferase insert mismatches and random nucleotides at intersection regions between D and J regions (4). Without homology between different D or J regions, it is impossible to bind them *in vitro*.

![Figure 1](https://static.igem.org/mediawiki/2021/d/d7/T--UNIZAR--Eng22.png)

Figure 2. *In vivo* recombination and generation of nanobodies. V (pink), D (blue) and J (green) fragments recombine to generate new nanobodies. CDRs are represented in grey. As it can be seen, CDR3 is located in the intersection between D and J fragments, whose sequence variability makes impossible *in vitro* recombination of V, D and J fragments as there is no constant region.

Therefore, we decided to tackle the design with **another strategy**. We discovered that nanobody sequences have some really well-preserved regions, with scarce variations even between different species. They are known as FR and are responsible for nanobody's 3D structure (1). Two of them are just between the CDRs regions, which makes them perfect to use as overhangs for Gibson assembly.

We decided to select nanobodies **from different origins** against GFP as a "proof of concept" because it is easy to quantify antigen as it is a naturally fluorescent protein (measurements at 510nm after excitation at 488nm), perfect for evaluating at the end of the process the efficiency and **enrichment** on our newly formed nanobodies.

With our **new strategy** in which different species' nanobodies are mixed, not only the CDRs are randomized but also the FR regions allowing us to obtain a **higher variety** of nanobodies as 3D structure directly affects CDRs structure and exposition. In this way, we will solve synthetic libraries limitation described above.

![Figure 1](https://static.igem.org/mediawiki/2021/d/db/T--UNIZAR--Eng15.png)

![](https://static.igem.org/mediawiki/2021/3/38/T--UNIZAR--Eng3.png)

## BUILD. What nanobodies and which regions are we going to use for our design?

We divided each nanobody into three fragments separating them by the **FR conserved regions**, keeping these regions at the ends. As each fragment contains one CDR, from now on we will refer to each fragment as CDR1, CDR2 and CDR3, respectively (although CDRs in fact are just a region and not the entire fragment). All our nanobody sequences were chosen from the online [Single Domain Antibody Database](http://www.sdab-db.ca/)  (5), except one, that was taken directly from Potsdam Bioware 2021 iGEM team part registry: BBa\_K929104 (6).

We first chose different antiGFP nanobodies from **4 different origins**: arabian camel (*Camelus dromedarius*), llama (*Llama glama*), alpaca (*Vicugna pacos*) and synthetic. The sequences' codons were adapted for expression in *E. coli* in order to improve the efficiency of the system.

The final nanobody regions we have designed can be found in  [Parts.](https://2021.igem.org/Team%3AUNIZAR/Parts)

We **compared all their sequences** in order to find the perfect complementary regions (FRs). In this way any CDR1 can bind to any CDR2 through *Gibson assembly* as they share the same sequence and any CDR2 can bind as well to any CDR3 as they share another sequence.

* CDR1-CDR2 assembly sequence: TGGTTCCGTCAGGCGCCGGGTAAA
* CDR1-CDR2 assembly sequence: TGGTTCCGTCAGGCGCCGGGTAAA

|  |  |
| --- | --- |
| ![Figure 4](https://static.igem.org/mediawiki/2021/e/ed/T--UNIZAR--Eng16.png) | ![Figure 5](https://static.igem.org/mediawiki/2021/8/89/T--UNIZAR--Proof3.png) |
| Figure 4. Nanobody sequence alignment with MAFFT in Benchling. Red dots determine regions with sequence differences between nanobodies. Two internal regions, labelled constant 1 and 2 (in blue) are completely conserved and thus useful for recombination (7). | Figure 5. Representation of recombination step. CDR1-CDR2 assembly sequence in blue and CDR2-CDR3 assembly sequence in pink. Each green fragment represents a nanobody fragment, with this technology fragments from different nanobodies recombine creating a whole new nanobdy. |

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/7/78/T--UNIZAR--Eng2.png)

## TEST. Do these regions recombine well and form new nanobodies?

Firstly, we successfully amplified by PCR each CDR fragment that was **synthesized** by IDT. In those PCRs we used these primers:

* CDR1 forward: ACCTCAGAATTCTATGGCAGACGTTC
* CDR1 reverse: TTTACCCGGCGCCTGACG
* CDR2 forward: TGGTTCCGTCAGGCGCC
* CDR2 reverse: GTCACGAGAGATGGTGAAACGACC
* CDR3 forward: GGTCGTTTCACCATCTCTCGTGAC
* CDR3 reverse: GCCGATCGACTTTGCTAAGCTTC

With enough quantity of CDRs we managed to assemble our CDRs into **recombinant nanobodies** composed by CDR1, CDR2 and CDR3. On our first approach, we used the CloneEZ PCR cloning kit with CDRs from one single Alpaca nanobody (nanobody A12). We succeeded in making the recombination:

![Figure 6](https://static.igem.org/mediawiki/2021/3/33/T--UNIZAR--Results2.png)

Figure 6. Electrophoresis of nanobodies PCR-amplified nanobodies (NBs). Negative controls are labelled as c-. A12 NB was obtained from Clone EZ assembly of three fragments: its CDR1, CDR2 and CDR3. Cloning was successful as its band after PCR amplification Cl-Ez corresponds to the A12 nanobody sequence, generated in vitro through the combination of its CDRs. The rest of bands correspond to complete chemically synthesized nanobodies, which act as positive controls for the PCR.

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/1/16/T--UNIZAR--Engin1.png)

## LEARN. What happened?

The **cloning efficiency** of the recombination product in a digested plasmid with restriction enzymes was very **low**, so trying another cloning strategy is necessary in order to get better results. We focused in finding a new approach that allowed us to continue with the working pipeline.

![Figure 1. Science, business, and law in</div>terrelation in technology transfer](https://static.igem.org/mediawiki/2021/1/16/T--UNIZAR--Engin1.png)

## IMPROVE. How can we increase the efficiency of the recombination process?

Since the two-step assembly was not successful we took another approach. We tried **direct Gibson Assembly cloning** by introducing in the CDR1 and CDR3 a homologous sequence with the plasmid sequence in the insertion site.

![Figure 7](https://static.igem.org/mediawiki/2021/1/1f/T--UNIZAR--Eng9.png)

Figure 7. Gibson Assembly design. The blue and red regions in the plasmid are also present in the new nanobody sequence as it can be introduced by PCR using long primers.

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/3/38/T--UNIZAR--Eng3.png)

## DESIGN. New primers for Gibson Assembly!

We designed two **new primers** that allowed us to introduce a sequence in the CDR1 and CDR3 for recombination with the plasmid. The new primer sequences are shown below (region complementary to the plasmid sequence appear in bold type).

* CDR1 forward with homology sequence: **GCCGCCGCGGCCGC**GAATTCTATGGCAGACGTTCAGCTGC
* CDR3 reverse with homology sequence: **TGTCGAGCGGCCGC**AAGCTTCTAGAAGAAACGGTAACCTG

![Figure 7](https://static.igem.org/mediawiki/2021/6/69/T--UNIZAR--Eng8.png)

Figure 8. introduction of homologous sequences in the 3' and 5' end of the recombined nanobodies by PCR.

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/7/78/T--UNIZAR--Eng2.png)

## TEST. Does this new approach provide better results?

After cloning by Gibson Assembly the recombinant nanobodies pool in a plasmid, in order **to know if the insert was well cloned**, we performed PCR with primers that hybridized outside the MCS. If a short amplified product (200 pb) was detected it means that the cloning didn't go really well, if a long product is detected (500 pb) the strategy worked. After analyzing the amplification products in an agarose gel we observed that the cloning problems persisted, unfortunately.

![Figure 9](https://static.igem.org/mediawiki/2021/2/2b/T--UNIZAR--Results6.png)

Figure 9. Agarose 0.8% electrophoresis gel. The 5th lane corresponds (without taking the marker into consideration) to amplification of the insert with the nanobodies inserted in it. A very weak line was detected at 500 pb while there is an intense one at 200 pb.

However we were able to randomly recombine the whole pool of sequences and obtain newly formed nanobodies, we were able to generate ***in vitro* libraries**! This is a huge step forward in the project results. In order to confirm the variability generated by this method, we wanted to sequence the new nanobody pool obtained [in collaboration with ARIA team from Barcelona](https://2021.igem.org/Team%3AUNIZAR/Partnership) . Although we were not able to send them a sample enough enriched in nanobody containing plasmids

![Figure 10](https://static.igem.org/mediawiki/2021/9/92/T--UNIZAR--Results3.png)

Figure 10. Electrophoresis of nanobody sequences in a 0,8% agarose gel. Second-line corresponds to the recombination product of all CDR sequences available.

# PLASMID DESIGN AND PURIFICATION

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/7/78/T--UNIZAR--Eng2.png)

## RESEARCH. What type of expression vector are we using for our nanobody library?

We wanted to have a system that allowed us to test the newly formed nanobodies in an easy way. We discovered that there are bacterial proteins called **intimins and autotransporters** that are used to display small proteins in the surface of bacteria such as *E. coli* (8). These proteins are currently being used to display nanobodies and other small proteins (9,10), so we decided that it could be a good expression strategy for our project.

![](https://static.igem.org/mediawiki/2021/3/38/T--UNIZAR--Eng3.png)

## DESIGN. Designing the cloning strategy

For the plasmid design, we asked pSEVA repository (11) and contacted [Dr. Esteban Martínez,](https://2021.igem.org/Team%3AUNIZAR/Human_Practices)  a researcher from Zaragoza that is currently working in the CNB. He sent us 3 plasmids from the **pSEVA repository**: pSEVA228-I, pSEVA238-I, pSEVA228-AT. They differed on its nanobody expression strategy: intimin-transported (228I, 238I) and autotransporter (228AT) and on their copy number, being low for 228I and medium for 238I. Both plasmids confer *E. coli* resistance against **kanamycin**.

![](https://static.igem.org/mediawiki/2021/a/ac/T--UNIZAR--Engineering.png)

Figure 11. pSEVA228-I and pSEVA228-AT maps. Both of the systems allow displaying nanobodies on the surface of bacteria when cloned in the MCS (multiple cloning site). The main difference lies in the MCS position, in pSEVA228-AT it is in 5' end of AT but in pSEVA228-Intimin it is in the 3' end.

 In order to achieve nanobody display, their sequence must be cloned in the **MCS** of the plasmids. We used the restriction site *Eco*RI in the 5' end and *Hind*III in the 3' end (Figure 7), these sites were introduced in the sequence by chemical synthesis.

![](https://static.igem.org/mediawiki/2021/2/2b/T--UNIZAR--Proof.png)

Figure 12. Representation of MCS of plasmids (blue). The restriction sites used are highlighted in yellow. Figure made in SnapGene.

It is **important** to design the sequence of the nanobody in order to **maintain the ORF in frame with the signal peptide** (*pelB*) included in the plasmid and the fusion protein that will allow membrane display. The signal peptide guides the system to the bacterial periplasm, and finally, the intimin (or the autotransporter) is located in the external membrane of *E. coli* exposing the nanobody. Taking this into consideration, the sequences were designed as follows.

![](https://static.igem.org/mediawiki/2021/b/b9/T--UNIZAR--Eng5.png)

Figure 13. Sequence of a consensus nanobody. Restriction sites for *Eco*RI and *Hind*III in purple, in by their sides there must be a nucleotidic tail to ensure that the restriction enzyme is able to cut within its site. In green, there are represented nucleotides that have been introduced in order to maintain the ORF and ensure good expression. The starting codon is highlighted in red and the STOP codon has been removed as it is included in the plasmid in the E-tag sequence in pSEVA228-I and pSEVA238-I and in the autotransporter sequence in pSEVA228-AT.

The plasmids also contain an E-tag that allows us to detect protein expression levels by using an anti-E-tag antibody in western and ELISA assays.

![](https://static.igem.org/mediawiki/2021/9/98/T--UNIZAR--Eng4.png)

Figure 14. Expression system based in intimins displaying the nanobody in the external membrane of *E. coli*.

![Figure 1. Science, business, and law interrelation in technology transfer](https://static.igem.org/mediawiki/2021/1/14/T--UNIZAR--Eng6.png)

## BUILD. What cloning technique will be used for nanobodies display?

Plasmids pSEVA228-I, pSEVA238-I and pSEVA228-AT were originally expressed in *E. coli* XL1-Blue and purified via **miniprep**. Afterward, they were digested using *Eco*RI and *Hind*III restriction enzymes in order to open the MCS. The recombinant nanobody DNA was then cloned into the plasmid and transformed into chemically competent *E. coli*.

![](https://static.igem.org/mediawiki/2021/7/78/T--UNIZAR--Eng2.png)

## TEST. First purification of pSEVA plasmids

Firstly, plasmid purification was evaluated through quantitative and qualitative methods: **nanodrop DNA concentration measurement** and visualization at **agarose gel electrophoresis**. We found that our purification was inefficient as the plasmid concentration we obtained was lower than expected for low-copy plasmids [(see results).](https://2021.igem.org/Team%3AUNIZAR/Results)

The lack of high plasmid concentrations influenced the rest of the experiments performed. Digestions, clonings, transformations and gel visualization weren´t optimal as very few plasmids was obtained and its quality differed in every extraction.

![](https://static.igem.org/mediawiki/2021/1/16/T--UNIZAR--Engin1.png)

## LEARN. What can we do now?

Doing **good plasmid purification is a critical step** in order to have good results in the next steps of the molecular design. In order to improve the low quantities of purified plasmid, we decided **to try different purification protocols miniprep kits**, but all of them were proven insufficient. As it seemed the miniprep kit was not a problem, we began to think that the issue could be the *E. coli* strain we were working with.

Moreover, we noticed that by **purifying directly from a plate colony**, we could obtain **higher concentrations** than from liquid culture. Consequently, we decided to prepare new glycerol stocks from the plates we obtained a greater amount of plasmid from.

![](https://static.igem.org/mediawiki/2021/3/38/T--UNIZAR--Eng3.png)

## DESIGN. Is it worth it to change the strain that keeps the plasmids?

Bearing in mind what we have learned from our previous experiences, we decided to transform our plasmid into a new *E. coli* expression strain: **DH5α as it lacks RecA so no homologous recombination between plasmids can occur** and endonucleases so plasmid degradation is difficult (12). This strain is usually used for plasmid keeping and purification.

![](https://static.igem.org/mediawiki/2021/1/16/T--UNIZAR--Engin1.png)

## BUILD. *E. coli* DH5α transformation and plasmid purification

This *E. coli* strain was transformed with the best previous plasmid purification by thermic shock with the three plasmids.

![](https://static.igem.org/mediawiki/2021/c/cc/T--UNIZAR--Proof9.png)

Figure 15. Representation of *E. coli* chemical transformation protocol and selection in plaque in presence of kanamycin.

![](https://static.igem.org/mediawiki/2021/7/78/T--UNIZAR--Eng2.png)

## TEST. Plasmid purification results

Unfortunately, plasmid concentration results remained below our expectations and complicated further work with the plasmids.

![](https://static.igem.org/mediawiki/2021/a/aa/T--UNIZAR--Eng14.png)

## LEARN. Asking for help is always a great option!

In sight of the problems we were having with plasmid purification, we contacted  [ARIA iGEM team](https://2021.igem.org/Team%3AUNIZAR/Partnership)  and [Dr. Esteban Martínez](https://2021.igem.org/Team%3AUNIZAR/Human_Practices)  in order to get their support in this step we were stuck. Thanks to the advice and protocols they provided us with, we were able to increase the  [plasmid concentration.](https://2021.igem.org/Team%3AUNIZAR/Results)

Now we are able to perform experiments with higher quality and efficiency as the plasmid is no longer an issue!

# References

1. Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013 Jun ;82:775–97.
2. IMGT Home page [Internet]. [cited 2021 Oct 10]. Available from: [http://www.imgt.org/](https://www.imgt.org/)
3. Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084–102.
4. Richard A. Goldsby, Thomas J. Kindt, Janis Kuby BAO. Kuby Immunology. 2002
5. Single Domain Antibody Database [Internet]. [cited 2021 Oct 10]. Available from: <http://www.sdab-db.ca/>
6. Part:BBa K929104 - parts.igem.org [Internet]. [cited 2021 Oct 10]. Available from: [http://parts.igem.org/wiki/index.php/Part:BBa\_K929104](http://parts.igem.org/wiki/index.php/Part%3ABBa_K929104)
7. Editor · Benchling [Internet]. [cited 2021 Oct 10]. Available from: <https://benchling.com/editor>
8. Dalbey RE, Kuhn A. Protein Traffic in Gram-negative bacteria - how exported and secreted proteins find their way. FEMS Microbiol Rev. 2012;36(6):1023–45.
9. Veiga E, De Lorenzo V, Fernández LA. Autotransporters as scaffolds for novel bacterial adhesins: Surface properties of Escherichia coli cells displaying Jun/Fos dimerization domains. J Bacteriol. 2003;185(18):5585–90.
10. Salema V, Fernández LÁ. Escherichia coli surface display for the selection of nanobodies. Microb Biotechnol. 2017;10(6):1468–84.
11. SEVA plasmids – Standard European Vector Architecture – : Your database and repository of standard, modular plasmid vectors for constructing complex bacterial phenotypes [Internet]. [cited 2021 Oct 11]. Available from: <http://seva-plasmids.com/>
12. NEB® 5-alpha Competent E. coli (Subcloning Efficiency) | NEB [Internet]. [cited 2021 Oct 19]. Available from: <https://www.neb.com/products/c2988-neb-5-alpha-competent-e-coli-subcloning-efficiency#Product> Information

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# Team:UNIZAR/Experiments

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### **Experiments**

# Protocols

Here, you can find protocols used by our team.

##### Competent cells

It allows the captation of free external DNA by bacteria

[Download](https://static.igem.org/mediawiki/2021/6/6e/T--UNIZAR--Prot1.pdf)

##### Plasmid purification

Extraction of the plasmid from the bacterial cytoplasm

[Download](https://static.igem.org/mediawiki/2021/d/d5/T--UNIZAR--Prot2.pdf)

##### Double digestion

For directional cloning two restiction enzymes are used

[Download](https://static.igem.org/mediawiki/2021/2/28/T--UNIZAR--Prot3.pdf)

##### DNA assembly

*In vitro* recombination of DNA fragments with homologous regions

[Download](https://static.igem.org/mediawiki/2021/9/96/T--UNIZAR--Prot4.pdf)

##### Transformation

Competent cells are able to capture free exteranl DNA such as plasmids

[Download](https://static.igem.org/mediawiki/2021/f/f0/T--UNIZAR--Prot5.pdf)

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# Team:UNIZAR/Results

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### **Results**

# NANOBODIES (NBs) SEQUENCE ANALYSIS

Analysis of NBs sequences revealed the existence of two constant regions between CDRs of different NBs. These regions are responsible for nanobodies' 3D structure and are perfect to be used for assembly. Thanks to these preserved regions, we were able to design independent nanobody fragments composed of CDR and overhanging regions, which are capable of overlapping and combining with any other CDR with the same overhanging region. These fragments were chemically synthesized and from now on we will refer to them as CDR1, CDR2 and CDR3, respectively, although they do not correspond exactly with the actual CDR [(see part collection).](https://2021.igem.org/Team%3AUNIZAR/Part_Collection)

![Figure 1](https://static.igem.org/mediawiki/2021/4/4b/T--UNIZAR--Results1.png)

Figure 1. Sequence allignment of different anti-GFP nanobodies from different species. Mismatches to the reference sequence are shown in red. Two regions with common sequences can be observed in the middle of every nanobody sequence.

# CDR ASSEMBLY

## A12 nanobody

We demonstrated that we were able to recombine a CDR1 with a CDR2 and a CDR3 to create an *in-vitro*-generated nanobody by overlapping their constant regions. This implies that our original design was successful and that the overlapping sequences we designed did work.

A12 nanobody's three fragments: CDR1, CDR2 and CDR3 were combined through CloneEZ PCR cloning kit. 1 µL of each CDR at 10 ng/µL were mixed with 1 µL of buffer, 1 µL of enzyme and 5 µL of nuclease free water. The solution was then kept 30 minutes at 22 ºC. A sample was taken in order to check through PCR amplification that the nanobody had been correctly assembled, using the complete NB A12 as positive control (fig. 2).

![Figure 2](https://static.igem.org/mediawiki/2021/3/33/T--UNIZAR--Results2.png)

Figure 2. Agarose gel electrophoresis showed that nanobody A12 was obtained from its CDRs through Clone EZ PCR cloning. Original NB A12 and CloneEZ product show a band for the same molecular weight.

## CDR pools

We demonstrated that we were able to generate nanobodies through recombination of three different pools of CDR. We combined all CDR1, CDR2 and CDR3 from different nanobodies, generating new ones.

In this case, CloneEZ PCR cloning DNA samples consisted on three mixtures of all CDR1, all CDR2 and all CDR3, respectively. Each pool at a final concentration of 10 DNA ng/µL. 1 µL of each mix was added to the assembling reaction with the CloneEZ PCR cloning kit.

Assembly was checked through PCR amplification of newly formed complete NBs as explained above. It was afterwards loaded in a agarose gel electrophoresis together with a secondary control of amplification: a NBs pool composed of NBs mixed in equal proportions (Figure 3).

![Figure 3](https://static.igem.org/mediawiki/2021/9/92/T--UNIZAR--Results3.png)

Figure 3. Agarose gel electrophoresis of PCR amplification of NB A12, the NBs pool and the assembly of the CDRs pool. PCR amplifications were developed at 5 different temperatures, from 58ºC to 62ºC.

## *Eco*RI + *Hind*III PLASMID DOUBLE DIGESTION

The next step consisted in cloning nanobodies' sequences into plasmids. In order to achieve this, we first needed to digest our purified plasmids with *EcoR*I and *Hind*>III restriction enzymes.

We intented to find the most appropiate digestion conditions testing two different buffers: Red and TANGO buffers from ThermoFisher and three digesting times: 1 h, 4 h and overnight. In addition, we checked the individual activity of both enzymes in the different tested conditions to ensure none of them could degrade the plasmid.

![Figure 4](https://static.igem.org/mediawiki/2021/a/a6/T--UNIZAR--Results4.png)

Figure 4. Visualization on agarose gel electrophoresis of the digested plasmids on different conditions. There is no sign of degradation in any of them although the band is barely visible in some single digestion samples.

As overnight digested plasmids were well digested and not degraded we decided to use overnight digestions in our future experiments to reduce as much as possible the amount of undigested plasmid.(Figure 4)

## PLASMID CLONING

After following the whole process of plasmid purification, digestion, insert cloning and transformation, we found out that most of our competent cells had no insert at all. Seeing the transformation results, we could concluded that many of the bacteria had been transformed with undigested plasmid with no insert (Figure 5).

This hypothesis was also confirmed by PCR amplification of the plasmid MCS. As it can be seen in Figure 6, two bands appear in the transformed cells samples. A lower one (lighter) corresponding to the undigested MCS (200 bp) and a series of higher ones (heavier) that could correspond to inserted nanobodies (≈500 bp). These ones are absent in the original plasmid control (228 AT).

![Figure 5](https://static.igem.org/mediawiki/2021/6/6a/T--UNIZAR--Results5.png)

Figure 5. Negative control of transformation with numerous grown colonies. This negative control was obtained by transforming digested, theoretically linear, plasmid directly into competent cells, with no insert. The rest of the transformation plates with insert had a non-significantly greater amount of colonies.

![Figure 6](https://static.igem.org/mediawiki/2021/2/2b/T--UNIZAR--Results6.png)

Figure 6. PCR amplification of liquid cultures of transformed competent cells. The amplified fragments correspond to the MCS, with or without insert, of the transformed plasmids. GeneRuler 100 bp DNA ladder was used.

## TROUBLESHOOTING

With the aim of dealing with the undigested plasmids, we thought that an effective solution could be purifying digested plasmid directly from its electrophoresis agarose gel band. Nevertheless, we were not able to follow this strategy since our original plasmid quantity was too low (Figure 7) to recover a significant amount of it from the gel. Consequently, we tried to increase plasmid purification efficiency.

## PLASMID PURIFICATION

Plasmids pSEVA228-I, pSEVA238-I and pSEVA228-AT were originally purified from liquid culture of *E. coli* XL1-Blue. As the plasmid concentration obtained was unsatisfactory (Figure 7), different miniprep kits were used. Similar results were obtained with all of them, indicating they were probably not the cause of the low plasmid concentration.

We decided to keep using the same miniprep kit and to change the plasmid expression strain to another well-known laboratory strain, DH5α, which is commonly used for transformation and plasmid expression. Even though there was a mild increase, the plasmid concentration results remained below our expectations.

To improve our results we asked for advice to other iGEM teams. We sent samples of our plasmids to UPF Barcelona iGEM with the aim of knowing if they could obtain higher concentrations [(see Partnership).](https://2021.igem.org/Team%3AUNIZAR/Partnership) Moreover, we asked  [Dr. Esteban Martinez](https://2021.igem.org/Team%3AUNIZAR/Human_Practices)  for guidance and he recommended as to elute different columns with the same buffer in order to get higher concentrations of plasmid.

![Figure 7](https://static.igem.org/mediawiki/2021/7/7e/T--UNIZAR--Results7.png)

Figure 7. Comparative results of plasmid concentration nanodrop measurements obtained from XL1 strain liquid culture, DH5α strain liquid culture and XL1 strain using Dr. Esteban Martinez's strategy. This strategy allowed us to work with a greater volume of liquid culture and so to obtain more plasmid.

Using homemade maxiprep for plasmid purification, we obtained DNA concentrations over 200 ng/µL or even 300 ng/µL. Unfortunately, when observed at agarose gel electrophoresis , these samples turned out to be degraded or contaminated with RNA (Figure 8).

![Figure 8](https://static.igem.org/mediawiki/2021/1/11/T--UNIZAR--Results8.png)

Figure 8. Agarose gel after electrophoresis of plasmid samples contaminated with RNA. Samples showed concentration values of 260.5 ng/µL, 266.6 ng/µL and 310.4 ng/µL, respectively. Over the RNA, the band of the plasmid could be seen but its quantity was too low for band purification.

## PLASMID PCR

Another strategy to deal with undigested plasmids was amplifying by PCR our purified plasmids except the MCS. In this way we would ensure to obtain linear plasmid, ready to be used for cloning with no need to improve plasmid purification results. Furthermore, PCR amplified plasmids would never close without the insert, securing no more bacterial growth in our transformation negative controls.

We wanted to find the most appropiate PCR conditions, so we assayed three different DNA polymerases: NEB Hifi, PrimeStar and MyTaq Bioline at three different annealing temperatures: 56 ºC, 58 ºC and 60 ºC and with our three plasmids: 228I, 238I and 228AT.

Only one of the conditions tested allowed linear plasmid amplification. However, such amplification was not reproducible on further attempts.

![Figure 9](https://static.igem.org/mediawiki/2021/1/14/T--UNIZAR--Results9.png)

Figure 9. Agarose gel electrophoresis showing the PCR results, with no positive results except one, that was proved to be not reproducible.

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