iGEM 2022 — Team barcelona-ub


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{% block title %}Engineering success{% endblock %}
{% block lead %}"Shall I refuse dinner because I do not fully understand the process of digestion?" Oliver Heaviside{% endblock %}
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#### INTRODUCTION

Throughout the development of our project, we have faced multiple obstacles that have forced us to look for other ways to achieve our objectives. We have had to go through various troubleshooting processes that, satisfyingly, have boosted our learning in several concepts and techniques. While doing so, we have performed a series of engineering cycles.

#### Assembly strategy

The first strategy that we conceived for the assembly of our constructs was based on the Golden Gate technique. For that, we designed our constructs as multiple fragments with sticky ends generated within a Golden Gate reaction to be assembled into the recipient pJUMP29-1A KanR Level 1 backbone. In order to save time and simplify the reactions, all the constructs were designed to be assembled in a level 1 backbone, avoiding the need of cloning them on level 0 backbones or further cloning them into level 2 ones (for more information see Contribution wiki). Furthermore, we designed the fragments in a modular fashion to take advantage and use the same common fragment for multiple constructions (CMV promoter, SV40 polyA).
Once we tested that the assembly of each of our constructs was successful in silico (analysis performed with benchling.com), we sent the fragments to be built by iGEM sponsors IDT and Twist Bioscience. Once in the wet lab, the experimental tests did not work as we expected: we only achieved the assembly of the constructs formed by a low number of fragments (1-2 fragments), whereas the assemblies of the ones formed by more fragments (4-6 fragments) were not achieved.

In that context, we have performed several engineering cycles to determine the proper Golden Gate conditions for each assembly. We tried out several conditions for the reaction, modifying both the number of cycles, the duration of each temperature, the moment of introducing each enzyme and the duration of the last ligation step. We even tried to cut and purify the open backbone beforehand to increase the ligation of the fragments. With that many experiments performed we learned that the proper conditions for our constructs depend on the number of fragments to be assembled in a single Golden Gate reaction.

However, after we achieved the ligation of most of our constructs, we made an awful discovery that explained the bad efficiency of our Golden Gate assemblies. We were performing the assemblies using BsmBI, which is in the reverse orientation! Instead, we should have used the BsaI sites, as they have a correct orientation that allows the ligation of the fragments while hindering the entry of the removed part of the backbone. The source of this problem in the design, apart from our inexpertise and lack of contact with experts, resides in the backbone's map sequence in the Part Registry. This sequence is incomplete! It lacks the BsaI sites and the GFP in between the main assembly site. Sadly, we did not realize this until when, upon request, we obtained the correct map sequence of the backbones.

![Scheme of golden gate cloning](https://static.igem.wiki/teams/4501/wiki/eng-suc1.jpg)

**Figure 1. Issue in the Golden Gate strategy.**(A) BsmBI assembly sites inside the pJUMP level 1 backbone in which we wrongly based our first design for pursuing the Golden Gate strategy. (B) Correct assembly sites in which we based our second design.

It was too late, but we re-designed our unsuccessfully assembled constructs to be divided into fragments for a BsaI assembly (for example BBa\_K4501021).

In parallel, given the technical difficulties for the assembly we resolved to re-design our assembly strategy and try the fusion PCR technique. For that, we asked Dr. Fernando Setién, an expert on the field, for guidance and general guidelines. Once we came up with the desired strategy, we designed several pairs of primers that we used to amplify and add the adapters required to the gene fragments. Once more, the test did not work and we tried several conditions for the fusion PCR, but none of them worked (see more on Results).

Curiously, at the end the constructs were successfully assembled by Golden Gate technique, so the fusion PCR strategy was discontinued.

#### pJUMP-based Lentiviral Transfer Plasmid

Our first design of the pJUMP-based transfer plasmid had some important limitations that restricted the potential versatility and amenability of the device. Namely, the 5’ module had some BsmBI and BsaI recognition sites that impeded the assembly on the main site. For that reason, the device was first conceived to be cloned sequentially: the 5’ module was to be cloned after the main assembly, which extended the procedure and made it more complicated and expensive. When we built and tested the device, we faced assembly difficulties and we realized that the pJUMP-based transfer plasmid was not optimal at all.

For that reason, we decided to re-design the 5’ and 3’ modules so they didn't incorporate BsmBI nor BsaI recognition sites. That way, we would be able to clone both of them previous to the main assembly reaction, shortening the procedure and reducing costs. Furthermore, we decided to incorporate a SV40 ori of replication downstream of the 3’ module, supplying the backbone with a powerful ori that could increase the transfection efficiency onto the HEK293T cell line.

![Scheme of golden gate cloning](https://static.igem.wiki/teams/4501/wiki/eng-suc2.png)

**Figure 2. Map sequence of the final version of the pJUMP-based lentiviral transfer plasmid.**

#### Validation of anti-myc shRNAs

One of our greatest engineering successes is the design of our own shRNA against myc. Using the VectorBuilder Wizard for shRNA generation, we designed a shRNA against the 1741-1761 region of myc, for which we constructed an expression cassette coupled to a GFP reporter. To that shRNA (BBa\_K4501018), we made two different variants that incorporated the C/D-box label for exosome loading: one containing the C/D-box after the 3’ end of shRNA (BBa\_K4501019) and another incorporating the C/D-box in substitution of the traditional loop (BBa\_K4501020) (Figure 3).

We managed to assemble all the constructs into a pJUMP backbone by Golden Gate assembly, and we performed a qPCR to assess myc expression (see Results webpage). In that, we obtained an almost 2-fold reduction of myc expression in Ramos cells treated with the shRNA, indicating its functionality and the success of the design.

![Scheme of golden gate cloning](https://static.igem.wiki/teams/4501/wiki/shrna-no-cd-box.png)

**Figure 3. Representation and stability of the different constructions of shRNA that we designed.**

#### His-tag-based affinity chromatography purification of exosomes

We also succeeded in developing our own way of purifying exosomes with only instruments that can be found in a normal laboratory. By using homemade column affinity chromatography with HisPurTM Ni-NTA resin (ThermoFisher, Batch protocol), we achieved purification (though with a low yield) of His-tagged exosomes (see Results webpage). However, further improvement of the system (p.e. stably transfecting with lentiviral vectors) could increase the yield of exosome purification.

![Scheme of golden gate cloning](https://static.igem.wiki/teams/4501/wiki/disseny-sense-t-tol-13.png)

**Figure 4. NTA results showing the obtained concentration of nanoparticles after ultracentrifugation or His-tag mediated affinity chromatography of the CD63\_His-tag part (BBa\_K4501013).**

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## EXPERIMENTS & PROTOCOLS

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## LAB NOTEBOOK

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{% block title %}Results{% endblock %}
{% block lead %}“If we knew what it was we were doing, it would not be called research, would it?”
Albert Einstein{% endblock %}
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Despite the overwhelming large extension of our planned project, we set out in the wet lab to achieve as many results as possible. Although our final purpose was not reached due to time limitations, we firmly believe that our evidence is enough to expect the system to work as predicted and to prove that our project is not only viable but functional.

## ACHIEVED RESULTS

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#### HEK293T CELL LINE IS SENSIBLE TO PUROMYCIN AND HYGROMYCIN AS INDICATED BY MTT ASSAYS MTT assay is a colorimetric assay used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity; thus, widely used to determine the antibiotic resistance of cellular lines. In our case, the assay was used to determine the most optimal **antibiotic concentration** to select the cells that incorporated the constructs, which included an antibiotic resistance gene. Initially, we prepared the same dilution bank for hygromycin and puromycin, although the second one turned out to be more damaging than expected for the HEK293T cell line and required the use of lower concentrations. Each well of the plate was cultured with the proper antibiotic and concentration of it for 7 days, during which the team performed a qualitative assay concerning the cells' growth inhibition. Moreover, on the last day of the assay, we performed a quantitative assay to obtain the inhibition curved. Results are shown in Figure 1 (Fig. 1.1 and Fig. 1.2) and Figure 2. The following concentrations were chosen due to their capacity to maintain the cell's viability during the first 48 hours, but to kill them after 7 days: * Hygromycin: 600 ug/uL * Puromycin: 0,4 ug/uL ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' one (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/1-1.png) Figure 1.1. Table summarizing the results of the MTT assay 'Quantification of Hygromycin'. Results show a correlation between the increase in antibiotic concentration and its capacity to inhibit cellular growth. The value marked in green corresponds to the Hygromycin concentration that maintains the cell's viability during the first 48 hours but kills them after 7 days. ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' one (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/1-2.png) Figure 1.2. Table summarizing the results of the MTT assay 'Quantification of Puromycin'. The value marked in green corresponds to the Hygromycin concentration that maintains the cell's viability during the first 48 hours but kills them after 7 days. ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0 %FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0 %FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/results-figure-3.jpg) Inhibition growth curve of Hygromycin and Puromycin. The values marked in green correspond to the optimal antibiotic concentrations for the MTT assay in our HEK293T cell line. EXOSOMES OF THE HEK293T CELL LINE ARE SUCCESSFULLY PURIFIED THROUGH LOW REVOLUTIONS ULTRACENTRIFUGATION The first step of our project was focused on assessing the default exosome production of the HEK293T cells, despite the existence of previous scientific data that proved it1, 2; establishing a baseline exosome production of the cells, and validating the exosome purification and collection methods. It must be pointed out that FBS medium, which is necessary for the survival and growth of the HEK293T cell line, contains exosomes and contaminants for its visualization; thus, needs to be removed and replaced by 0% FBS medium before the analysis of the cells. To assess the impact of the medium removal on the survival and growth of the cells, we studied two different conditions: changing the complete medium for 0% FBS 48 hours or 24 hours before collecting the exosomes. The ultracentrifugation protocol (see Protocols) was followed to isolate the exosomes of the cells. To visualize and quantify the exosomes we used the Nanoparticle Tracking Analysis (NTA) with the generous help of Marc Nicolau (Dr. Hernando Del Portillo’s group). The results of both conditions (Fig.3.) indicate a significant exosome enrichment in the samples resulting from the ultracentrifugation protocol. The '24h with 0% FBS' condition showed a higher purity outcome in the 100-300 nm sized particle fraction; which, in addition to its simpler procedure, made it elective for further experiments. ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0 %FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/fig2-res.jpg) Figure 3. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies. GOLDEN GATE TECHNIQUE ALLOWS THE ASSEMBLY OF SEVERAL GENE FRAGMENTS INTO LEVEL 1 pJUMP BACKBONE IN A SINGLE REACTION AND EVEN IN THE REVERSE ORIENTATION The second step of our project was focused on selecting and applying an assembly strategy for our constructions. Considering the amenability of the method, the existence of commercial sponsors, and the availability of suitable plasmid backbones for it in the Distribution Kit, we chose the Golden Gate technique. Our team divided each of the composite Parts (see Parts webpage) into a different number of fragments, which were adapted to be assembled into the pJUMP29-1A KanR Level 1 pUC backbone [(http://parts.igem.org/Part:BBa\_J428353)](http://parts.igem.org/Part%3ABBa_J428353) with a single reaction. The economization of the number of reactions allowed us to avoid unnecessary intermediate cloning steps and to obtain the construction through a simple step. Nonetheless, we were misled by the sequence map of the backbone in the Part Registry and we designed our fragments to fit in a BsmBI assembly, which generated a restriction site with reverse orientation in the backbone (see Figure 1 of Engineering success webpage). Since the recognition sites of the BsmBI were not being removed from the backbone, the enzyme was cutting and ligating them in each cycle, thus greatly reducing the efficiency of the reaction. To obtain a smooth ligation, we should have employed the BsaI restriction site, but this one was not present in the sequence map of the Part Registry [(http://parts.igem.org/Part:BBa\_J428353)](http://parts.igem.org/Part%3ABBa_J428353). Our team was not aware of this mistake until upon request it was shown the correct sequence map of the pJUMP backbone. Despite this huge setback and trying out dozens of different conditions for the assembly reactions, we were able to successfully clone several of our constructs (Figure 4). Hence, we only had to re-design some of the constructs for a BsaI-based assembly. The BsmBI-based assembly protocol that turned out to be the most successful consisted of cutting the constructs with the BsmBI restriction enzyme, purifying the linearized backbone, and afterward performing several cycles of 37ºC-16ºC. To visualize and quantify the exosomes we used the Nanoparticle Tracking Analysis (NTA) with the generous help of Marc Nicolau (Dr. Hernando Del Portillo’s group). The results of both conditions (Fig.3.) indicate a significant exosome enrichment in the samples resulting from the ultracentrifugation protocol. The '24h with 0% FBS' condition showed a higher purity outcome in the 100-300 nm sized particle fraction; which, in addition to its simpler procedure, made it elective for further experiments. ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/results-figure-4.jpg) Figure 4. Sequencing results that verify the successful assembly of several constructs into our pJUMP backbone. FUSION PCR FOR THE LIGATION OF GENE FRAGMENTS IS NOT ACHIEVED In parallel to the inefficient Golden Gate assemblies, our team decided to try the Fusion PCR technique with the CD63\_His-tag construct (BBa\_K4501013). We designed primers that incorporated overlapping sequences (consisting of 15-22 bp homology regions with the same melting temperature) to the extremes of the different parts, in order to fuse the consecutive ones in each cycle of the reaction. Two different backbones and restriction enzymes (BsmBI or EcoRI/BamHI) were used to assess this approach. However, despite trying four different conditions, any of them allowed us to obtain the desired construct (data not shown). ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/results-figure-5.jpg) Figure 5. Successful amplification of the fragments used in the assembly of the BBa\_K4501013 construct through Fusion PCR. THE 3’ MODULE ON THE pJUMP BACKBONE IS NOT SUFFICIENT FOR SUCCESSFUL LENTIVIRUS PRODUCTION Due to time limitations, our team decided to set up an experiment to verify the ability of our pJUMP-based transfer plasmid to produce a lentiviral vector, in parallel with the development of the constructs. However, considering the presence of BsmBI and BsaI recognition sites in the 5' module, which impeded the assembly of the main site, we had to opt for sequential cloning: 1. Cloning of the 3' module, 2. Cloning of the main assembly, and 3. Cloning of the 5' module. The previous design hindered the cloning of the 5' module on time. However, we tried to produce lentivirus with the pJUMP containing exclusively the 3' module, which could be assessed by GFP expression. The lentivirus were produced with 10 ug of the transfer plasmid, 7.5 ug of pPAX.2, and 2.5 ug of pMD2.G with the TurboFect reagent, in the HEK293T packaging cell line; and then used to infect wild-type HEK293T cells. Unfortunately, no GFP fluorescence was observed (data not shown). Taking the previous into account, our team decided to re-design the 5’ and 3’ modules to exclude the BsaI and BsmBi restriction sites; and, thus, allow its pre-assembly on the pJUMP backbone. An SV40 origin of replication was further added to improve the transfection outcome of the backbone in the HEK293T cell line (see Engineering Success webpage). TRANSIENT TRANSFECTION WITH THE CD63\_His-tag-L7Ae ALLOWS LOW-YIELD AFFINITY CHROMATOGRAPHY PURIFICATION OF THE EXOSOMES After accomplishing the assembly of CD63\_His-tag and CD63\_His-tag-L7Ae, our team decided to set up an experiment to verify the ability of the HEK293T cell line to produce exosomes that could be purified by simple His-tag affinity chromatography. We transfected HEK293T cells by electroporation, either with 5 ug of CD63\_His-tag or 5 ug of CD63\_His-tag-L7Ae. After 24 hours, the media was changed to 0% FBS and after 24 hours more the supernatant was collected to purify the exosomes. The expression of the reporter of the construct (RFP) was assessed by FACS and exhibited very low values (Figure 6A). Our team followed the ultracentrifugation protocol and affinity chromatography protocol as described. For the last one, 7 mL of cleared supernatant was passed through a falcon tube containing 2 mL of HisPurTM Ni-NTA resin (ThermoFisher) and the sample was bound according to the instructions of the manufacturer. ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/results-figure-6.jpg) Figure 6. (A) Flow cytometry plots that show the RFP expression levels. (B) Results that summarize the concentration of nanoparticles determined with the ultracentrifuge protocol and the His-tag mediated affinity chromatography protocol. (C) NTA plots that show the size profile of the purified particles. The eluted fractions resulting from the ultracentrifugation protocol and the affinity chromatography protocol were assessed by NTA analysis. The fraction obtained through the affinity chromatography protocol displayed a concentration two orders of magnitude lower than the one obtained through the ultracentrifugation protocol. However, the values of the first one were considered significantly high in comparison with the background signal (approximately 104 particles/mL); and, thus, this protocol proved to be able to retain some of the particles of interest. Moreover, particle size analysis confirmed the presence of fewer contaminants (particles above 200 nm) in the fraction obtained with the affinity chromatography protocol, in comparison to that one of the ultracentrifugation protocol (Figure 6C). Nonetheless, it must be pointed out that the unbound and the eluted fraction of the experiment displayed similar amounts of particles (Figure 6C), leading us to think that the protocol is not optimized. Results of our improved CD63\_His-tag-L7Ae are equivalent (data shown in the Part Registry, BBa\_K4501014). Conjointly, these results may suggest that the His-tag-mediated affinity chromatography protocol is viable and, when optimized, could be useful to purify exosomes more efficiently. The observed low efficiency might be due to a low electroporation efficiency of the cells. In the future, the lentiviral transfection with CD63\_His-tag and CD63\_His-tag-L7Ae will be optimized until the detection of high levels of reporter fluorescence. Additionally, it must be highlighted that our team also managed to visualize exosomes in a FACS Canto cytometer through exosome binding to DynabeadsTM His-tag isolation (Invitrogen) (data available upon request). OUR DESIGNED shRNA INDUCES DOWNREGULATION OF MYC WITH A SINGLE TRANSIENT TRANSFECTION The final step of our project was focused on assessing the capacity of our shRNA to downregulate myc expression. We transiently transfected HEK293T and Ramos cell lines with three different versions of the shRNA (BBa\_K4501018, BBa\_K4501019, and BBa\_K4501020) and, after 72 hours, the cells were collected to extract their total RNA. The total RNA was used to create a total cDNA population and perform a qPCR to asses myc expression. Actin-Beta and GADPH genes were used as housekeeping references. The Ramos condition, in which our team only tested the raw version of the shRNA in expectancy of lower efficiency, showed almost a 2-fold reduction in the expression of myc (Figure 7A); hence, proving its functionality. However, little to no effect was observed on all of the HEK293T conditions, perhaps due to their conditioning in 0% FBS medium for 24 hours, which could have masked the effect of the shRNA. Interestingly, almost no GFP expression was detected by flow cytometry in any of the conditions (Figure 7B); in line with the RFP expression results of the CD63 constructs. Considering the proper functionality of the parts, this event may be indicative of an issue with the reporter expression (e.g. in the bicistronic construction, in the promoter, or by proximity with the main expression cassette). Further attempts with lentiviral vectors will be needed to elucidate the source of this conflict. ![Figure 2. Representation of the NTA results showing the exosome yield of the different conditions. The concentration (y-axis) and the particle size (x-axis) show a prominent peak from 100 to 300 nm that corresponds to the population of exosomes. The '24h with 0% FBS' condition (left image) seems to be more enriched with exosomes than the '48h with 0% FBS' condition (right image). The increase in the total number of particles in the second condition might be due to the contamination of the sample with apoptotic bodies.](https://static.igem.wiki/teams/4501/wiki/results/results-figure-7.jpg) Figure 7. The designed shRNA downregulates myc expression in the Ramos' Burkitt lymphoma cell line. (A) Representation of the qPCR myc expression fold change in the Ramos and the HEK293T cell lines. The total RNA was used to create a total cDNA population. Actin-Beta and GADPH genes were used as housekeeping references. (B) GFP expression levels of the HEK293T cell line transfected with the different shRNA constructs. Bibliography 1. Zhu X, Badawi M, Pomeroy S, Sutaria DS, Xie Z, Baek A, Jiang J, Elgamal OA, Mo X, Perle K, Chalmers J, Schmittgen TD, Phelps MA. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J Extracell Vesicles. 2017 Jun 6;6(1):1324730. doi: [10.1080/20013078.2017.1324730.](https://pubmed.ncbi.nlm.nih.gov/28717420/) PMID: 28717420; PMCID: PMC5505007 2. Vilanova-Perez T, Jones C, Balint S, Dragovic R, L Dustin M, Yeste M, Coward K. Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro. Nanomedicine (Lond). 2020 Aug;15(20):1965-1980. doi: [10.2217/nnm-2020-0056](https://pubmed.ncbi.nlm.nih.gov/32794431/) . Epub 2020 Aug 14. PMID: 32794431. {% endblock %}