iGEM 2021 — Team BOKU-Vienna

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# Team:BOKU-Vienna/Notebook

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# Notebook

Cloning

Scaffolds

Cell culture

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### About us

We are a team of students from different Viennese universities enthusiastic about advances and innovation in synthetic biology.

### Contact us

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# Team:BOKU-Vienna/Engineering

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# Design & Engineering

Our project was quite complex with many different facets and potential pitfalls involved. As a result, our progressions during the iGEM engineering cycle were numerous and of quite different nature.

Thus, we structured this section - as we structured all our lab work - into the topics “Cloning” and “Scaffold”.

![](https://static.igem.org/mediawiki/2021/5/5b/T--BOKU-Vienna--Engineering_Icon_01.svg)

Original design by vectorjuice / [Freepik](http://www.freepik.com)

Cloning

Scaffold

# Assembly of our Type-II-S backbone

### Design

As we planned to assemble our constructs in *E.coli* and then switch to *L.plantarum* for the actual expression, we needed a fitting vector. The vector needed to work in both host organisms and match the Type-II-S assembly system. We started by using the plasmid pSIP403, which contained five sequences that would be identified as illegal restriction sites in the Type-II-S system, which we referred to as "illegal sites" in the following paragraphs.

![](https://static.igem.org/mediawiki/2021/e/e7/T--BOKU-Vienna--Engineering_Cloning_01.jpeg)

Our approach included the decomposition of the plasmid pSIP403 into five pieces, because of the five illegal sites in the original plasmid. Hence, we designed 10 primers: one forward and one reverse primer for each of the five illegal sites. These primers would anneal a few basepairs (bp) downstream of the illegal site. The design of the primers includes a 5’ overhang upstream of the annealing site which does not match the template sequence. With this we excluded the plasmid section in which the illegal site resides. When running a PCR, the primers with the overlap will get incorporated in the newly synthesized DNA, resulting in a growing amount of DNA constructs devoid of the illegal sites. To obtain the respective five constructs, an Agarose gel purification of the PCR products was performed including further purification. For the re-assembly of the single constructs, a Golden Gate assembly approach was taken, yielding the full backbone 3 (BB3). The overhang of the primers was constructed to carry a *BsaI* restriction site as well as a fusion site, ensuring the correct order of constructs during the assembling.
This sophisticated primer design allowed us to use the pSIP403 to create our novel BB3. Additionally, the reporter gene GuS was removed and instead, we decided to use amilCP, a blue chromoprotein, as a reporter.

![](https://static.igem.org/mediawiki/2021/3/33/T--BOKU-Vienna--Engineering_Cloning_02.jpeg)

pSIP403 served as template for the constructs one to four. For construct five, we needed to use a vector carrying the amilCP gene, and we decided for the pET 30a amilCP plasmid.

### Build and test

In the first run, a classical PCR was performed, followed by a preparative gel. The gel yielded good results for construct one, construct two and construct four. After gel purification and DNA sequencing, the identity of the respective constructs was confirmed. Construct three and construct five did not work under the originally defined conditions.

### Learn and re-design

We decided to test if we had more success by slightly deviating the primer annealing temperature. We faced the problem of having very faint bands on the stained Agarose gels and multiple bands not matching the desired size. We troubleshot our problem and decided to use a touchdown PCR for our next approach since this kind of PCR regimen can reduce the number of unspecific PCR products. During the annealing step of each cycle, the temperature was decreased by 1°C per cycle. We started with a higher temperature (72 °C) than the calculated melting temperature (Tm) and gradually decreased the temperature.

### Build and test

The touchdown PCR yielded a positive result for construct five, but the amount of PCR product was not sufficient for further processing. Construct three still refused to be synthesized. We tried further PCR runs with small modifications (different elongation temperatures and different amounts of template DNA) but still, no positive result could be achieved.

### Learn and re-design

Because of the unique overhang-containing structure of our primers, we came up with the idea to split the PCR program into two different stages: the first 10 cycles were done using a lower annealing temperature well adapted for the Tm of the primers allowing for more specific binding since the overhang of the designed primer will not anneal when the original template is used. After five to 10 cycles one can increase the annealing temperature since the newly synthesized DNA will act as a new template, enabling the primers to anneal fully. The remaining 20-35 cycles are performed with the higher annealing temperature matching the new Tm.

### Build and test

With this method we were able to increase the signal and yield a sufficient amount of construct five.

### Learn and re-design

Nevertheless, construct three still failed. This time we decided to try a two-step PCR. This means the annealing temperature is set at the same value as the elongation temperature of 72 °C resulting in two steps - denaturation, and elongation. The idea of this approach is to get the maximum specificity. For this to work, we had to design new primers with a Tm matching the elongation temperature. We achieved this by increasing the length of the primers downstream.

### Build and test

This method finally yielded a signal for construct three, enabling us to finally assemble the raw BB3.
The primers for construct five had another addition. Apart from the *BsaI* restriction site, they also included a *BbsI* restriction site. This enabled us to insert different linkers, including fusion sites, into the raw BB3 making it suitable for the insertion of up to four expression cassettes in one single BB3. (BBa\_K3855401)

# Backbone 2 expression cassette

### Design

We designed the respective sequences for inserts, promoters, and terminators then codon-optimized them for the expression in *Lactobacillus*. In combination with the process of codon optimization, we cured our sequences from illegal sites. After finishing the design, we ordered the g-blocks from IDT (Integrated DNA Technologies) and cloned them into backbone 1 (BB1).

### Build and test

This first step of our cloning process did not pose a challenge and the building blocks for our expression cassette were ready. But when we tried to assemble them into backbone 2 (BB2), we failed to get positive results. There was hardly any growth observable and the colonies picked showed no insert when sequenced.

### Learn and re-design

Since promotor, the gene of interest (our enzymes), and terminator are assembled into a single plasmid during this step of the cloning process, the result was a functional expression cassette. As a consequence, the enzymes are expressed and translated into the catalytically active enzymes, causing various effects in the expression host including a high metabolic burden. We assumed that our promotors are too strong and thus pose a very high metabolic burden on the transformed clones. Therefore, we stepped back and used weaker promotors.

### Build and test

The weaker promotors chosen are P4 and P5 and have been reported to be up to 10 times weaker than P1, P2, and P3. We repeated the assembly as well as the transformation. As a result, we were rewarded with several clones which were processed, purified, and sent to sequencing.

### Learn and re-design

All clones turned out to contain a frameshift mutation. One example is shown below. The left-hand side shows the sequences of our three clones aligned with the expected sequence. A frameshift is seen at the first base pair of the enzyme sequence. On the upper right, the original sequence and the according translated protein sequences are shown, with the clone version containing a one-bp frameshift mutation in the coding sequence below. The mutation significantly alters the protein sequence, as it causes a shift in the open reading frame. During the first engineering cycle, the few clones we obtained, also showed mutations rendering the enzyme unfunctional. This indicated a reason, other than the metabolic burden, causing the complications.

![](https://static.igem.org/mediawiki/2021/2/22/T--BOKU-Vienna--Engineering_Cloning_03.jpeg)

Even though we found no hint towards the origin of this problem in the literature, we had to assume that our enzymes are toxic to *E.coli* in some way, as only clones with a functioning mutation survived.
Since the translated enzymes contained a secretion signal for *Lactobacillus*, we expected fewer complications as soon as we would reach *Lactobacillus* as an expression host. Therefore, we designed an approach that would allow us to circumvent cloning the BB2 in *E.coli*.

### Build and test

Cloning the plasmid into *E.coli* after the assembly has two main purposes: Selection of functioning plasmids and multiplication of the plasmid DNA. In order to avoid *E.coli* during the cloning process, we designed a way to perform these steps artificially.
We did so by running a PCR to replicate the plasmid DNA. Then, we isolated the right plasmids via a quantitative gel. Further, the constructs were purified and the DNA content was quantified.

### Learn and re-design

Apparently, this approach did not yield sufficient amounts of plasmid DNA to perform a successful transformation in *Lactobacillus* since the results of the transformation were all negative. We compared the documented amount of plasmid DNA needed for a successful transformation with our yield and were off by more than one power of ten.
Thus, we went back to the drawing board and decided to use an inducible promoter.

### Build and test

Using the inducible promotor ORF (BBa\_K3855006) yielded positive clones in *E.coli* with no mutation. Thus, we were able to progress further with our project. The assembly of the BB3 constructs containing the ORF promoter succeeded in *Lactobacillus* at the first try for almost all constructs. Finally, we achieved our goal of cloning the fructan-degrading enzyme cassette into *Lactobacillus*.

![](https://static.igem.org/mediawiki/2021/8/88/T--BOKU-Vienna--Engineering_Cloning_04.jpeg)

# Synthesis of pepNTT

### Design

One should be prepared that IDT will not be able to synthesize certain DNA sequences with a too high complexity value. This was the case for our second terminator: pepNTT. We came up with two different approaches to synthesize the desired sequence. The vector pSIP403 carries the pepNTT terminator. Therefore, we designed two primers to amplify pepNTT using pSIP403 as a template sequence. Since we could not guarantee the success of this approach, our second idea was to assemble primers into the full pepNTT sequence. In our case, the sequence was divided into five pieces of approximately 55 bp in size. For the 3’ -> 5’ direction, the five pieces were shifted approximately 20 bp to create large enough overlapping sequences to ensure a reasonable Tm for the annealing of the single pieces to each other. The terminal area of the construct not covered by the overlapping sequence is covered by a smaller primer. The graphic shown illustrates the arrangement of the primers forming the pepNTT terminator.

![](https://static.igem.org/mediawiki/2021/8/85/T--BOKU-Vienna--Engineering_Cloning_05.jpeg)

### Build and test

To get the full sequence, we tested different assembly approaches. The assembly is performed in a classical thermocycler and the following program was the most effective:
98 °C for 20 seconds followed by a decrease of 1 °C per 15 seconds until 59 °C. Then follows a quick cooldown to 8 °C for two minutes.
Subsequently, a preparative gel including the respective purification steps was performed to obtain the purified construct. Both approaches yielded not only correct pepNTT terminator constructs but also a sufficient DNA concentration.

# Cellulose sulfate

### Design

For the scaffold, which in our case is a polyelectrolyte complex, we needed two strongly opposingly charged polymers. The positively charged polymer is polyDADMAC which is commercially available. The negatively charged component is water-soluble cellulose sulfate which is not commercially available and thus needed to be synthesized.

### Build

The synthesis of the cellulose sulfate was carried out under the supervision of Dr. Hubert Hetteger. With his help, we sulfated microcrystalline cellulose wit the modification of a lower amount of SO3-Pyridin (1 mol equivalent) according to his method (See Protocol SSP in PolyDADMAC).

### Test

After the successful synthesis of cellulose sulfate, we tried to form a polyelectrolyte complex capsule by dropping the cellulose sulfate solution into the polyDADMAC solution using a syringe. Unfortunately, the capsule did not form since the cellulose sulfate solution started to spread in the solution and precipitated.

### Learn

We discovered that the cellulose sulfate synthesized from the microcrystalline cellulose was of too low molecular weight to form a capsule. Thus, we needed cellulose of much higher molecular weight.

### Design

The candidates were spruce sulfite pulp (short: SSP) and a cotton linter, which have a higher molecular weight than the microcrystalline cellulose.

### Build

The same sulfation method as described above was used for the synthesis of the respective sulfared, negatively charged polyelectrolyte. Only the raw material, the unmodified cellulose, has been changed.

### Test

We discovered that the cellulose sulfate we tried to synthesize using different raw materials was not water-soluble. There were also a few undissolved cellulose fibers in the otherwise clear solution.

### Learn

The problem was that the synthesized cellulose was neither completely dissolved in the DMA (N,N-Dimethylacetamid) solution nor homogeneously distributed. Thus, we added more LiCl, since the molecular weight of the cellulose derived from raw materials than microcrystalline cellulose is higher. A further problem we encountered, were the hygroscopic properties of SO3-pyridine and LiCl. Since the sulfating reaction needs to take place in the total absence of water, we needed to work with dry substances. The water would react with SO3 and form sulfuric acid.

### Design

We now knew that we had to work water-free as much as possible and tried to completely dissolve the cellulose fibers.

### Build

To keep our substances dry, we used drying beads and a vacuum dryer. Fresh chemicals were closed with parafilm to protect them from humidity. We also dissolved the celluloses in an already prepared DMA/LiCl solution, the rest of the method remained unchanged.

### Test

There were no longer any undissolved fibers, but the synthesized cellulose sulfate was still not water-soluble when we tried to solve it in distilled water and diluted NaOH.([4](https://doi.org/10.1016/j.eurpolymj.2019.07.030))

### Learn

Water-soluble cellulose is achieved when the substitution degree with sulfate is high enough and homogeneous.

### Design

To achieve a higher degree of substitution with sulfate, higher concentration of SO3-Pyridin was used.

### Build

The amount of SO3-Pyridin was increased. Instead of 1mol equivalent, three to eight equivalents were used for further experiments.

### Test

The cellulose sulfate made from spruce sulfite pulp and cotton linters using more SO3-Pyridin and working as water-free as possible was water-soluble. We dissolved the cellulose sulfate and dripped it in the polyDADMAC solution. Capsules were formed, and the cellulose sulfate synthesized from spruce sulfite pulp and cotton linters showed similar results in the FTIR spectrum (see [Notebook in the Scaffolds section](https://2021.igem.org/Team%3ABOKU-Vienna/Notebook)). But since some material loss of the cotton linters at the filtration step happened, we decided to work mostly with the spruce sulfite pulp.
The capsule stability was tested by first placing the empty capsules in distilled water overnight. At the next day, it was still intact and the bottle containing the capsule was put on a slow-moving shaker for five hours (see [Notebook in the Scaffolds section](https://2021.igem.org/Team%3ABOKU-Vienna/Notebook)). The formed capsules were left in distilled water over several days and the stable ones were also shaken slowly for a few hours on a shaker.

### Learn

Water-soluble anionic cellulose sulfate which formed scaffolds when mixed carefully with cationic polyDADMAC was produced. But there was not enough time left to fine-tune solution concentrations, thereby also adjusting the pore size, and other modifications to enhance the properties.

# Thiolation and Mucoadhesivity

### Design

One of the main functions of our scaffold - besides retaining the cells - is its mucoadhesivity. In order to achieve it, we decided to use a method called thiolation - meaning the addition of thiol groups. These can then form disulfide bridges with cysteine residues of proteins lining the gut mucosa and thus, would allow our scaffold to adhere to the mucosal surface.
We initially planned to confer mucoadhesivity via direct thiolation of cellulose sulfate. Since cellulose sulfate is not commercially available, we wanted to carry out a synthesis of our cellulose sulfate using microcrystalline cellulose as raw material and then in a second step, thiolation would be achieved using thiourea and a microwave (see [protocol section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments)).

### Build

The thiolation was carried out in the lab of Prof. Andreas Bernkop-Schnürch at the University of Innsbruck. He was kind enough to share his methods and expertise on thiolated polymers with us. Thiolation of our own cellulose sulfate derived from microcrystalline cellulose was done according to a published method ([1](https://doi.org/10.3390/pharmaceutics13060854)) using a microwave, but we implemented changes to the original protocol.([2](https://doi.org/10.1016/j.ijpharm.2020.119040))

### Test and learn

We tested the success of the thiolation and, thereby mucoadhesion, performing two tests called "Ellman’s test" and "disulfide bridge test" with 5,5-di-thio-bis-(nitro-benzoic acid).([3](https://doi.org/10.1016/j.ijpharm.2020.119040)) Unfortunately, we saw that something was wrong with our cellulose sulfate. After the thiolation treatment, it had become insoluble in water.
We learned that microwave irradiation led to the desulfation of cellulose sulfate. This was very unfortunate, since the negative charges of the sulfate are needed to form the polyelectrolyte complex that holds our bio container together.

### Re-design

So instead of thiolating cellulose sulfate directly, we had to come up with an alternative solution. We needed a material that would not disturb the polyelectrolyte complex formation while being biocompatible and susceptible to our thiolation method.
We chose to add another polysaccharide, chitosan, which we wanted to thiolate prior to adding it. Chitosan is the second most prevalent naturally occurring polysaccharide after cellulose and it is present in the shells of crustaceans, insect exoskeletons, and in some fungi. Our idea was to add the thiolated chitosan directly into the polyDADMAC (polydiallyldimethylammoniumchlorid) solution that could then be used to encapsulate the cells suspended in a cellulose sulfate solution. Thanks to its positive charge, chitosan should be well compatible with the cationic polyDADMAC solution and not interfere with its ability to form the polyelectrolyte complex. Also, with this approach, the chitosan should be located on the outside of the capsule and thus, be able to easily interact with the surface of the gut mucosa and form the desired disulfide bridges.

### Build and test

We thiolated chitosan in the laboratory of Prof. Bernkop-Schnürch using the same microwaving method as described above for the initial experiement and confirmed the successful thiolation using the Ellman’s test and disulfide bridge tests.
Back home in Vienna, we started our first encapsulation assays. In the beginning, we focused on assessing capsule stability using empty capsules without cells. There we saw that despite the capsules being slightly opaquer than without chitosan addition, they showed good stability over several days.
Next, we went on to do experiments using living *E.coli* cells and tested whether they could escape the bio container. There we saw that capsule formation was successful but over time more cells inside the capsule died than in capsules only composed of cellulose sulfate and polyDADMAC.

### Learn

We figured out that this might be due to the fact, that chitosan has certain anti-bacterial properties. This pointed into the direction that it still influenced the cells inside the container, although most of the chitosan must be located on the outside of the capsule since we need the sulfide of the thiolated chitosan outside to form disulfide bridges with the cysteine of the mucosa.
After this, we unfortunately ran out of time and were not able to conduct further experiments.

### Outlook

To optimize the capsule properties and allow better cell survival, further experiments using different concentrations of thiolated chitosan, polyDADMAC, and cell densities for encapsulation could be done in order to fine-tune the conditions and achieve optimal results.

# References

(1) Grosso, R., & de-Paz, M. V. (2021). Thiolated-Polymer-Based Nanoparticles as an Avant-Garde Approach for Anticancer Therapies-Reviewing Thiomers from Chitosan and Hyaluronic Acid. Pharmaceutics, 13(6), 854. <https://doi.org/10.3390/pharmaceutics13060854>

(2) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. <https://doi.org/10.1016/j.ijpharm.2020.119040>

(3) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. <https://doi.org/10.1016/j.ijpharm.2020.119040>

(4) Muhitdinov, B., Heinze, T., Turaev, A., Koschella, A., & Normakhamatov, N., (2019). Homogenous synthesis of sodium cellulose sulfates with regulable low and high degree of substitutions with SO3/Py in N,N-dimethylacetamide/LiCl. European Polymer Journal, 119, 181-188. <https://doi.org/10.1016/j.eurpolymj.2019.07.030>

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# Experiments

Cloning

Cell culture

Scaffolds

# Experiments

![](https://static.igem.org/mediawiki/2021/8/88/T--BOKU-Vienna--experiments_flow_chart.svg)

## GG assembly

Golden Gate Cloning is one of the easiest cloning methods as it provides a fast one-step reaction which leaves no cloning sites in the final construct,
cutting out unnecessary base pairs, therefore making it irreversible.
The restriction enzymes cut off the fusion sites of inserts and backbones, leaving matching overhangs to fuse. Typically, promotors, coding sequences and
terminators are purchased as gBlocks with the appropriate fusion sites and are then individually cloned into the first backbone, backbone 1.
Afterwards, promotors, coding sequences and terminators are fused into backbone 2. In the last step, multiple promotor-coding sequence-terminator blocks can be
cloned into a single final backbone.

![](https://static.igem.org/mediawiki/2021/3/3c/T--BOKU-Vienna--experiments_backbones.jpeg)

Fusion itself is done using a Thermocycler, often overnight. The assembly mix contains the inserts, backbones, ATP, DNA ligase, buffer, and water.
Following the assembly, DNA is transformed into competent cells (e.g., using heat shocking or electroporation).
Backbones contain an antibiotic resistance and cells are plated on media with respective antibiotics for selection.
After incubation the grown colonies can be picked, and DNA is extracted using a Miniprep system. Identity of the plasmid and inserts can be confirmed
via sequencing.Thereafter, the next cloning step may be performed by using the extracted and sequenced DNA as insert.

In silico planning of inserts must be done carefully. Appropriate fusion sites are needed, and sequences need to be optimized to not contain unwanted cutting sites (e.g., having a restriction site used in one of the ensembles inside of the coding sequence).However, one is rewarded with an easy, efficient, and reliable cloning system.

# Protocols

## GG assembly

The gBlocks were designed by our research team and ordered from IDT. Enzymes, buffers as well as the T4-Ligase were purchased from New England Biolabs. The backbones and ATP were kindly obtained from our supervisors.

### Vecors & Inserts

|  |  |
| --- | --- |
| Backbone 1\_Fusion site 1-2 | Promotors |
| Backbone 1\_Fusion site 2-3 | Enzymes |
| Backbone 1\_Fusion site 3-4 | Terminator |
| Backbone 2\_Fusion site A-B | Promotor + Enzyme 1 + Terminator |
| Backbone 2\_Fusion site B-C | Promotor + Enzyme 2 + Terminator |
| Backbone 2\_Fusion site C-D | Promotor + Enzyme 3 + Terminator |
| Backbone 3\_Fusion site A-B | P + E1 + T |
| Backbone 3\_Fusion site A-C | P + E1 + T & P + E2 + T |
| Backbone 3\_Fusion site A-D | P + E1 + T & P + E2 + T & P + E3 + T |

### Rescrition enzymes

|  |
| --- |
| Backbone 1 for BsaI |
| Backbone 2 for BbsI |
| Backbone 3 for BsaI |

### Reaction mix

|  |  |
| --- | --- |
| Total reaction volume | 20 µL |
| Vector | 1 µL |
| Fragment | 1 µL each |
| Restriction enzyme | 1 µL |
| CutSmart buffer 10x | 2 µL |
| ATP 10x | 2 µL |
| T4-Ligase (1:10 diluted 40 U) | 2.5 µL |
| RO-H2O | ad 20 µL |

All is added into a PCR tube. Enzymes are added last and kept in cooling racks until placed in Thermocycler.

### Golden Gate assembly program for Thermocycler: LONG

|  |  |  |  |
| --- | --- | --- | --- |
| Cutting | 37 °C | 5 min | 45 cycles |
| Ligation of sticky ends | 16 °C | 2:30 min |
| Fragment linearization | 50 °C | 5 min |
| Enzyme deactivation | 80 °C | 10 min |
| Cool down | 25 °C | 15 min |

### Golden Gate assembly program for Thermocycler: SHORT

|  |  |  |  |
| --- | --- | --- | --- |
| Cutting | 37 °C | 5 min | 40 cycles |
| Ligation of sticky ends | 16 °C | 2:30 min |
| Fragment linerarization | 50 °C | 5 min |
| Enzyme deactivation | 80 °C | 10 min |
| Cool down | 25 °C | 15 min |

Afterwards a post-digest is performed to degrade any backbones without inserts. For this 0.5 µL of the appropriate restriction enzyme is added
to each reaction tube and placed in the Thermocycler.

### Post digest program for Thermocycler

|  |  |  |
| --- | --- | --- |
| Cutting | 37 °C | 30 min |
| Enzyme deactivation | 65 °C | 20 min |
| Cool down | 25 °C | 1 s |

## Transformation

### Preparation of chemically competent *E.coli*

*E.coli* strain DH10B is inoculated from a glycerol stock onto an LB Agar plate. The inoculum is streaked on the plate using an inoculation loop to
obtain individual colonies. The plate is incubated overnight at 37 °C.
A flask with 10 mL of LB medium is inoculated with a single colony and incubated overnight in a shaker-incubator (37 °C, shaking 180 rpm).
The following day, 2 mL of this culture is transferred to a 2 L shake flask containing 200 mL LB medium and incubated for about 2.5 hours until OD600
reaches 0.6. It is checked continuously, first after 1.5 hours. Blank water (or LB medium) is used before every measurement and the cell mixture is not diluted.
The cells are cooled down on ice for 10 min and then pelleted in a centrifuge for 5 min at 4500 rpm (4000 × g) at 4 °C. The cells are resuspended in 80 mL volume
of ice-cold TFB1. The centrifugation step is repeated. The pellet is resuspended in 8 mL of ice-cold TFB2.
The cells are aliquoted 100 μL per tube and stored at −80 °C until used.

### Transformation in *E.coli*

1. Frozen chemically component cells (100 µL per tube) are thawed on ice.
2. 10 µL of GG product is added to the cells, cells and DNA are then gently mixed.
3. Let the cells-DNA-Mix stand on ice for 30 min.
4. Cells and DNA-mix are heat shocked for 90 s at 42 °C (exact time noted).
5. The cells are allowed to recover on ice for 5 min.
6. 1 mL of LB Medium without antibiotics is added and the cells are incubated at 37 °C for:
   * 90 min (if Kanamycin is used for backbone 1)
   * 30 min(if Ampicillin is used for backbone 2)
   * 60 min(if Erythromycin is used for backbone 3)
7. Cells are being distributed with a Drigalski spatula on the Agar plates at different concentrations:
   * 1 µL (add 90 µL LB Medium to the plate first)
   * 10 µL (add 90 µL LB Medium to the plate first)
   * 100 µL
   * Rest (centrifuge, discard all but 100µL of supernatant, resuspend and add 100 µL to plate)
8. The plates incubated at 37 °C overnight.

### Preparation of electrocompetent *L.plantarum*

1. Electrocompetent *L.plantarum* strain NC8 is always freshly prepared before a transformation.
2. A pre-culture is prepared the day before in 20 mL MRS-medium with a pH of 6.2 + 2 % glucose and placed in an anaerobic jar at 30 °C, 180rpm overnight.
3. The next day OD600 is measured, and it is calculated how much of the pre-culture needs to be added to another flask with 500 mL of MRS-medium +
   2 % glucose + 1 % glycine to equal an OD600 of 0.3.
4. The main culture is then grown at 30 °C, 0 rpm until OD600 of 0.6 is measured.
5. The culture is then centrifuged 10 min at 4000 rpm and 4 °C. All buffers are kept on ice throughout.
6. The pellet is suspended in 100 mL washing buffer and washed 5 times, each centrifugation step is 10min at 4000rpm and 4 °C.
7. The pellet is then resuspended in 1 mL of electroporation buffer and the OD600 is measured.

### Transformation in *L.plantarum*:

1. 80 µL of the electrocompetent cells are mixed with 20 µL DNA.
2. Electroporation is done at 2500 V, 200 Ω, 25 µF in 4 mm cuvettes à 6250 V/cm
3. Afterwards the cells are diluted with 900 µL MRS-medium + 2 % glucose + 0.3 M sucrose and incubated at 30 °C for 3h and 180 rpm.
4. The cells are plated on MRS Agar + 2 % glucose + 2.5 µg/mL erythromycin plates with concentrations of 50, 100, 200, 300 µL
   and incubated at 30 °C for 3 to 5 days.

## Colony PCR

### Reaction mix

|  |  |
| --- | --- |
| Total reaction volume | 10 µL |
| AccuStart II PCR SuperMix | 5 µL | *(hot-start polymerase)* |
| Forward sequencing primer | 1 µL |
| Backward sequencing primer | 1 µL |
| RO-H2O + picked colony | 3 µL |

All is added into a PCR tube and placed in the Thermocycler.

### PCR amplification program for Cycler

|  |  |  |
| --- | --- | --- |
| Disintegration & Activation | 94 °C | 5 min |
| 94 °C | 20 s | 35 cycles |
| 63 °C | 25 s |
| 68 °C | 2:10 min |
| Cool down | 25 °C | 2 s |

Promising clones are selected via agarose gel electrophoresis (1 % agarose gel, 130 V, 1 h) and an overnight culture is prepared for minipreps.

## PCR amplification (instead of transformation of BB3 in *E.coli*)

### Reaction mix

|  |  |
| --- | --- |
| Total reaction volume | 50 µL |
| DNA template | 5 µL |
| AccuStart II PCR SuperMix | 25 µL |
| Forward sequencing primer | 5 µL |
| Backward sequencing primer | 5 µL |
| RO-H2O | 10 µL |

All is added into a PCR tube and placed in the Thermocycler.

### PCR amplification program for Cycler

|  |  |  |
| --- | --- | --- |
| Disintegration & Activation | 94 °C | 5 min |
| 94 °C | 20 s | 35 cycles |
| 63 °C | 25 s |
| 68 °C | 2:10 min |
| Cool down | 25 °C | 2 s |

Promising clones are selected via a preparative agarose gel electrophoresis (1 % or 1.5 % agarose gel, 130 V, 1 h),
the appropriate bands are cut out and purified. The concentration is measured via Nanodrop, and the clones are sequenced for confirmation.

## PCR for construction of BB3

### Reaction mix

|  |  |
| --- | --- |
| Total reaction volume | 50 µL |
| DNA template | 5 µL |
| Q5® High-Fidelity 2X Master Mix | 25 µL |
| Forward sequencing primer | 2.5 µL |
| Backward sequencing primer | 2.5 µL |
| Nuclease free RO-H2O | 15 µL |

All is added into a PCR tube and placed in the Thermocycler.

### Elongation Temp + 25 s Annealing Temp:

|  |  |
| --- | --- |
| Construct 1, 2 & 3 | 50 sec + 25 sec = 75 sec |
| Construct4 & 5 | 24 sec + 25 sec = 49 sec |

### PCR amplification program for Cycler for construct c1-3 and c4-5:

|  |  |  |
| --- | --- | --- |
| Denaturation | 98 °C | 30 s |
| 98 °C | 10 s | 35 cycles |
| 72 °C | 75s/49s |
| 72 °C | 10 min |
| Cool down | 10 °C | 2 min |

Promising clones are selected via a preparative agarose gel electrophoresis (1 % or 1.5 % agarose gel, 130 V, 1 h), the appropriate bands are cut out and purified.
The concentration is measured via Nanodrop, and the clones are sequenced for confirmation.

## Miniprep & Gel Purification

According to protocol of the following Kits:

Miniprep for *E. coli*

Monarch® Plasmid Miniprep Kit by New England Biolabs
Hi Yield® Plasmid Mini Kit by SLG

Deviations:

* 2 mL culture used
* Extra step for sequencing done
* Eluted twice with 50 µL of 50 °C warm 10 mM TRIS

Deviations from MIDI prep of L. diolivrans for Mini prep of *L.plantarum*:

* 5 mL culture, harvest: 3200 rpm, 30 min
* Add 0.4 mL 5M LiCl (6.36 g/30 mL) - (2 mL 10M LiCl stock)
* Incubation: 37 °C, 180 rpm, 20 min
* Transfer mixture into 2 mL Eppis and centrifuge at 13000 rpm for 2 min
* Dissolve pellet in 1 mL AD
* Add 1 mL AD
* Centrifuge at 3200 rpm, 10 min
* Add Prep Mix (1 g Lysozyme + 500 µL RNAseA + 200 µL Mutanolysin-stock dissolved in 50 mL P1) and incubate at 37 °C, 180 rpm, 50 min
* Continue according to the blue protocol unit step 4
* Add 5 mL P1
* Add 10 mL P2
* Add 10 mL P3
* Continue according to the grey protocol from step 6 on
* Centrifugation
* Add TRIS and incubate at RT o/n

### Gel purification

ReliaPrep™ DNA Clean-Up and Concentration System by Promega
Wizard® SV Gel and PCR Clean-Up System by Promega

Deviations:

* Eluted twice with 15 µL of 50 °C warm 10 mM TRIS

## Restriction digest

### Reaction mix

|  |  |
| --- | --- |
| Total reaction volume | 10 µL |
| DNA (Miniprep) | 600 ng diluted with water to equal 20 µL |
| Enzyme 1 | 0.5 µL |
| Enzyme 2 (if necessary) | 0.5 µL |
| Buffer | 2 µL |

Samples are put on 37 °C for 1 h. Promising clones are selected via agarose gel electrophoresis (1 % agarose gel, 130 V, 1 h) and
subsequently confirmed via sequencing.

## Media

### LB medium & LB Agar for *E.coli*

|  |  |
| --- | --- |
| Peptone Hy-Soy | 40.00 g +/- 1.00 g |
| Yeast extract | 20.00 g +/- 0.50 g |
| NaCl | 20.00 g +/- 0.50 g |
| RO-H2O | ad 4000.00 g |

* Adjust pH to 7.4 - 7.6 with 4N or 8N NaOH
* Bottle 500 g in 500 mL Schott bottles
* Sterilize by autoclaving for 20 minutes at 121°C
* For LB Agar add 10.00 g +/- 0.25 g of Agar-Agar per 500mL Schott bottle

### MRS medium & MRS Agar for *L. plantarum*:

|  |  |
| --- | --- |
| RO-H2O | 2000.00 g +/- 50.00 g |
| Casein peptone | 40.00 g +/- 1.00 g |
| Meat extract | 40.00 g +/- 1.00 g |
| Yeast extract | 20.00 g +/- 0.50 g |
| Tween 80 | 4.00 g +/- 0.10 g |
| K2HPO4 | 8.00 g +/- 0.20 g |
| Sodium acetate | 20.00 g +/- 0.50 g |
| Tri-sodium citrate dihydrate | 10.40 g +/- 0.26 g |
| (NH4)2HPO4 | 4.68 g +/- 0.12 g |
| MgSO4 + 7 H2O | 0.80 g +/- 0.02 g |
| MnSO4 +H2O | 0.20 g +/- 0.01 g |
| RO-H2O | ad 3600.00 g |

* Adjust pH to 5.7 – 6.2 with HCl
* Bottle 500 g in 500 mL Schott bottles
* Sterilize by autoclaving for 20 minutes at 121°C
* Before use: Add 50 mL of 10 x carbon source (e.g., 10x glucose, 10x glycerol) per bottle
* for MRS Agar add 10.00 g +/- 0.25 g of agar-agar per 500mL Schott bottle

### Buffers for *L. plantarum* electroporation:

* Washing buffer
   0.3 M sucrose
* Electroporation buffer
   272 mM sucrose
   7 mM sodium phosphate
* Resuspension buffer
   MRS with a pH of 6.2 + 2 % glucose
   0.3 M sucrose

# Experiments

Since preclinical and clinical trials were out of scope for the iGEM competition, we were looking for a different way to test the biocompatibility of our scaffold.
We got in touch with Professor Peter Ertl from the Cell Chip Group at the Technical University of Vienna, who suggested that his organ-on-a-chip technologies might
be a good alternative, as they can mimic membrane barriers and fluidic systems which play an important role in the way how drugs interact with the body.
He gave us the opportunity to use his lab and receive guidance from Doris Roth for our experiments. She, however, advised us against using a gut-on-a-chip for several
reasons: One of the most important being that it can take several months until one gets a working and inoculated chip. Additionally, organs-on-a-chip are too small
to test a macroscopic capsule. In the end, we settled on going the classic route of an in vitro cytotoxicity test with Caco-2 cells and, optionally,
a transwell test to see if the polymer can pass through membranes.

Cytotoxicity tests in cell culture are well established and commonly used for the assessment of the biocompatibility of all kinds of materials. Since our capsule would only get in contact with intestinal membranes, the Cell Chip Group recommended Caco-2, an immortalized human colorectal adenocarcinoma cell line. This cell line is widely used as an in vitro model for the mucosa of the small intestine and used for the Cell Chip Group’s gut-on-a-chip.

To test our scaffold, we decided to do the resazurin or MTT assay, which is one of the most popular and widely used cell viability tests. It is an excellent way to assess the toxicity of a material in vitro. The assay is based on the reduction of oxidized non-fluorescent blue resazurin to a red fluorescent dye (resorufin) in cellular mitochondria. Therefore, a fluorescent signal indicates live cells. The proper seeding density must be adjusted to the respective cell line. Changes of incubation times with the test material as well as with the resazurin solution strongly affect the results, so they should be chosen wisely. Four hours of incubation with the resazurin solution are often regarded as ideal.

Cells used for assays should be active and healthy cultures (generally, they should be split at least twice to make sure they have recovered from being frozen) before using them in an assay. They should also not have been passaged too often, since at some point, genomic abnormalities can occur, or they start to grow more slowly.

Since our test material, chitosan, is not easily soluble in water at neutral pH but highly soluble in an acidic solution, we decided to decrease the pH to 6.3, which should be tolerated by Caco-2 cells. Here, we planned to test both the well-characterized chitosan, as well as our thiolated chitosan. Additionally, we thought about applying a fine suspension of powdered, lyophilized chitosan and thiolated chitosan, in case we would not manage to dissolve the polymers. Adding the whole capsule is no option since this might kill the cells or the material would get overgrown, making the results unusable. We chose to test a concentration of 0.25 % (w/v) since higher concentrations would likely be too viscous.

# Protocols

## Caco-2 - Thawing

### Introduction

Thawing of frozen Caco-2 stocks for culture

### Materials

* T25 flask
* 10 mL Caco-2 medium (total)
  + 80 % DMEM
  + 20 % FCS
  + 1 % streptomycin/penicillin (added on top)

### Procedure

Thawing:

1. Preheat medium to 37 °C.
2. Quickly thaw cells in your hand.
3. Add 4-5 mL preheated medium to the flask and add the cells from the cryovial. Gently mix by pipetting up and down.
4. Place the flask in the incubator. Criss-cross to distribute the cells evenly.

Change of Medium:

1. Place the flask in the incubator. Criss-cross to distribute the cells evenly.

## Caco-2 - Maintenance

### Introduction

Medium needs to be changed on Monday, Wednesday, and Friday. At 80-90 % confluency, cells need to be split (see protocol "Caco-2 - Splitting").

### Materials

Caco-2 medium

* 80 % DMEM
* 20 % FCS
* 1 % streptomycin/penicillin (added on top)

### Procedure

Medium exchange:

1. Preheat fresh medium to 37 °C.
2. Remove old medium from the side opposite to the cells as to not accidentally detach cells.
3. Add fresh medium, again at the opposite site.
   * For T25 flasks: 4-5 mL
   * For T75 flasks: 15 mL
4. Place flask back in the incubator.

## Caco-2 - Splitting (Passage)

### Introduction

If the cells are at 80-90 % confluency, they need to be split. The splitting ratio depends on how fast we want our cells to be ready for splitting again.
The usual ratio is 1:3 for Caco-2 cells. This means that 1/3 of the cells are transferred into the new flasks. Several flasks can be inoculated as needed.

### Materials

For T25 flasks:

* ~10 mL Caco-2 medium
* 2 mL trypsin
* 10-15 mL PBS (without calcium)

For T75 flasks:

* ~25 mL Caco-2 medium
* 3 mL trypsin
* 20-30 mL PBS (without calcium)

### Procedure

T25 flasks:

1. Preheat medium, PBS and trypsin to 37 °C.
2. Remove old medium.
3. Gently wash 2-3 x with 4 mL PBS to remove all remaining medium.
4. Add 2 mL trypsin.
5. Incubate for max. 5 min at 37°C until cells are detached.
6. Stop the reaction with 3 mL medium, washing down cells from the flask surface and transfer them into a Falcon tube.
7. Count cells if needed.
8. Centrifuge cells for 5 min at 1500 rpm.
9. Remove supernatant and resuspend cells in fresh medium.
10. Split cells 1:3 or as needed into new flasks to a total volume of 4-5 mL per flask. Don't forget to write down the new passage number.

T75 flasks:

1. Preheat medium, PBS and trypsin to 37 °C.
2. Remove old medium.
3. Gently wash 2-3 x with 10 mL PBS to remove all remaining medium.
4. Add 3 mL trypsin.
5. Incubate for max. 5 min at 37 °C until cells are detached.
6. Stop the reaction with 7 mL medium, washing down cells from the flask surface and transfer them into a Falcon tube.
7. Count cells if needed
8. Centrifuge cells for 5 min at 1500 rpm.
9. Remove supernatant and resuspend cells in fresh medium.
10. Split cells 1:3 or as needed into new flasks to a total volume of 15 mL per flask. Don't forget to write down the new passage number.

## Caco-2 - Seeding

### Introduction

Seeding of Caco-2 cells into 96-well plates for live/dead stainings to test biocompatibility or transwells for barrier experiments.
Cells should have been split twice before they are used for an experiment and be grown in a T75 flask to ensure that there are enough cells for the experiment
which are healthy and growing well.

### Materials

* Caco-2 medium
  + 80 % DMEM
  + 20 % FCS
  + 1 % streptomycin/penicillin (added on top)
* Trypsin
  + PBS (without calcium)
  + 96-well plates OR 24-well plates and transwells

### Procedure

Cell Detachment:

1. Cells should have been split at least twice before they are used for an experiment.
2. Preheat medium, PBS and trypsin to 37 °C.
3. Remove old medium.
4. Gently wash 2-3 x with 10 mL PBS to remove all medium.
5. Add 3 mL trypsin.
6. Incubate for max. 5 min until cells are detached.
7. Stop the reaction with 7 mL medium, washing down the cells from the flask surface.

Adjusting cell density:

1. Count cells and calculate dilution. Each well should contain 8000 cells; each well holds 200 µL volume.
2. Centrifuge cells for 5 min at 1500 rpm.
3. Remove supernatant and resuspend cell pellet in fresh, preheated medium to adjust cell density to 8000 cells/200 µL.

Seeding - 96 well plates:

1. Seed 200 µL cell suspension per well, leaving the outer rows empty or filling them with PBS to reduce evaporation.
2. Incubate at 37 °C.
3. Change medium after 4 h.
4. Grow overnight before starting the experiment.

Seeding - Transwell plates:

1. Add transwells to each well, leaving the top and bottom row empty or filling them with PBS to reduce evaporation.
2. Outer well: 400-500 µL
3. Inner well: 200 µL
4. Adjust outer surface level to avoid hydrostatic pressure.
5. Incubate at 37 °C.
6. Change medium after 4 h.

## Resazurin assay for cytotoxicity testing on Caco-2 cells

Unfortunately, due to limited time in the wet lab this experiment was not done. However, we decided to include this protocol in our Wiki to potentially
help future iGEM-Teams:

The resazurin or MTT assay is one of the most popular and widely used cell viability tests. It is an excellent way to assess the toxicity of a material in vitro.
The assay is based on the reduction of oxidized non-fluorescent blue resazurin to a red fluorescent dye (resorufin) in cellular mitochondria. Therefore, a
fluorescent signal indicates live cells. The proper seeding density must be adjusted to the respective cell line. Changes of incubation times with the test
material as well as with the resazurin solution strongly affect the results, so they should be chosen wisely. Four hours of incubation with the resazurin solution
are often regarded as ideal.

Cells used for assays should be in active culture and healthy (generally, they should be split at least twice to make sure they have recovered from being frozen)
before using them in an assay. They should also not have been passaged too often, since at some point, genomic abnormalities can occur, or they start to grow more
slowly.

Since our test material, chitosan, is not easily soluble in water at neutral pH but highly soluble in an acidic solution, we decided to decrease the pH to 6.3,
which should be tolerated by Caco-2 cells. Here, we planned to test both the well-characterized chitosan, as well as our thiolated chitosan.
Additionally, we thought about applying a fine suspension of powdered, lyophilized chitosan and thiolated chitosan, in case we would not manage to dissolve the polymers.
Adding the whole capsule is no option, since this might kill the cells or the material would get overgrown, making the results unusable. We chose to test a
concentration of 0.25 % (w/v) since higher concentrations would likely be too viscous.

* are seeded in a 96-well plate at a density of 2 x 104 cells per well in a final volume of 200 µL Caco-2 culture medium (see Seeding protocol).
* The cells containing the seeded cells are stored in the incubator (37 °C, 5 % CO2) until they have created a monolayer. This should take 24 h to max. 2 days.
* The samples are prepared at a final concentration of 0.25 % (w/v) in Caco-2 culture medium.
* Cells are washed 2-3 times with 100 µL PBS before test solutions are applied at a volume of 100 µL. The same volume of Caco-2 medium is applied to serve as a positive control,
  and Triton X 100 at a concentration of 4 % is added as a negative control. All samples are done at least in triplicates.
* The cells are again incubated for 24 h.
* Following the 24h of incubation, test and control solutions are removed, and after washing the cells twice with PBS, 100 µL of a 5 % (w/v) resazurin solution is applied.
* Cells are incubated for 4 h.
* The fluorescence intensity of each well is determined at an excitation wavelength of 540 nm and an emission wavelength of 590 nm using a microplate reader.
* Cell viability is calculated according to the following equation:

  Cell viability [%] = (intensity of sample – intensity of negative control)/(intensity of positive control – intensity of negative control) \* 100

# Experiments Our scaffold is one of the main parts of our project Friendzyme. Its main roles are: 1. Acting as a biocontainer 2. Providing mucadhesion 3. Protection of the cells against harsh conditions The biocontainer The biocontainer is an essential part of our biosafety concept which ensures that the GMOs we want to use do not proliferate freely and thus pose a threat to the patient or the environment. Our scaffold is designed in a way to keep the modified Lactobacillus separate from other bacteria in the gut. The pore size is chosen in a range that our Lactobacillus cannot escape through the mesh, but small molecules and secreted proteins can freely diffuse in and out. The modified lactobacilli are living off the nutrients provided by the intestinal lumen and in return secrete fructan-degrading enzymes that exit the scaffold and decompose the fructo-oligosaccharides before they can cause symptoms in the lower intestine (see Protocols: Enzyme Escape, and Cell Escape) Mucoadhesion The surface of our scaffold is designed to be mucoadhesive by the addition of thiolated chitosan to the polymer solution that forms the scaffold. It will stick to the mucosa of the gut after passing through the stomach by forming disulfide bonds with the mucosa, which contains a lot of cysteines and thus is abundantly interlinked by this type of strong, covalent bond. Its mucoadhesive properties will allow the scaffold to stay attached to the surface of the intestine and release the secrete there for a prolonged timespan. By that, we hope to create a novel system that - compared to classical enzyme therapy - will be based on living cells that continuously provide a constant supply of fructan-degrading enzymes and thus eliminate the need for frequent intake of medication (see Protocol: Thiolation). Protection It has also been shown that a scaffold made from cellulose sulfate and poly-DADMAC can protect cells from the aggressive environment of the gastric fluid. Although it is planned that administration of our scaffold particles will happen in the form of a coated pill this feature could potentially be beneficial. Our approach Our scaffold is composed of two biocompatible polymer solutions that upon mixing instantly form a polyelectrolyte complex due to their charge. The concept is based on the product “Cell in a Box” (Austrianova) and was modified in order to additionally achieve mucoadhesion. The cells are suspended in a solution containing negatively charged cellulose sulfate, one of the three main components of our biocontainer. Due to time restraints, we did not finish fine-tuning the synthesized cellulose sulfate and used for further tests the cellulose sulfate from the Cell-in-a-Box kit (see Protocol: Synthesis of Cellulose Sulfate). On the surface of the droplet where the two solutions come in contact the polyelectrolyte-complex forms spontaneously, yielding a stable gel-like shell with a certain mesh size, and the bacteria on the inside of the droplet are encapsulated within it. This solution is then dripped into a bath that contains positively charged Polydiallyldimethylammonium chloride (polyDADMAC), as well as thiolated chitosan to provide the mucoadhesive properties (see Protocol: own solution). ![](https://static.igem.org/mediawiki/2021/c/c7/T--BOKU-Vienna--experiments_1.png) Safety considerations The overall size of our scaffold capsules is considerably larger than the mucus mesh network spacing (100-200 nm). Thus, it will not be able to penetrate the mucus layer. References Gunzburg, W. H., Aung, M. M., Toa, P., Ng, S., Read, E., Tan, W. J., Brandtner, E. M., Dangerfield, J., & Salmons, B. (2020). Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulphate encapsulation. Microbial cell factories, 19(1), 216. <https://doi.org/10.1186/s12934-020-01465-3> Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. <https://doi.org/10.1016/j.ijpharm.2020.119040> ProtocolsEnzyme escapeMaterials * Bovine Serum Albumin (BSA), Sigma-Aldrich * Nanodrop * PBS * Austrianova Cell-in-a-Box® Kit * 12 eprouvettes * 3 ampoules Method 1. Prepare 12 eprouvettes with 3 mL kit 2 solution (dilute like described in package insert) 2. Prepare 3 ampoules with 150 µL PBS each 3. Add 2.5 mg of BSA to 120 µL kit 1 solution 4. Vortex 5. One drop in each eprouvette 6. Remove capsules after 10 min and put 5 capsules in each ampoule * Place ampoules on shaker (low speed) at room temperature 7. Take samples of 10 µL supernatant of each ampoule at every timepoint * t = 0 h; 0.5 h; 1 h; 3 h; 24 h 8. Measure samples with Nanodrop (measurement for protein A280) * Measure every sample 3x (3x2 µL) * Concentration should increase 9. Calibration line concentrations: * 0.0625 mg/mL; 0.125 mg/mL; 0.25 mg/mL; 0.5mg/mL; 1.0mg/mL; 1.4mg/mL * Blank: supernatant of empty capsule in 150 µL PBS Cell escape assayPurpose The purpose of this assay is to demonstrate the functionality of the polymeric scaffold. It can be used to assess whether the pore size is adequate to retain the cells on the inside of the capsule and prevent them from escaping into the surrounding medium. Material * Austrianova Cell-in-a-Box® kit * 1x Phosphate Buffered Saline (PBS) * Glass eprouvettes * LB-Agar plates (with antibiotic added if necessary) * Small glass tubes (we used HPLC tubes) * 1 mL syringes (Braun Sterican®) * Blunt-ended syringe needle, diameter 0.4 mm (Braun) * Small metal spatula 1. Experiment set-up * Prepare 15 eprouvettes each containing 3 mL kit solution 2 (dilute according to manual) * Prepare three glass tubes containing 100 µL PBS/capsule (final volume depends on how many capsules should be put into the tube) 2. Preparation of the cell solution: 1. To Spike 150 µL of kit 1 with 2\*108 *E.coli* cells/mL * Measure the OD of the cell culture * Calculate the right dilution for *E.coli* and centrifuge the cells for 7 minutes * Remove the supernatant and add the 150 µL of solution 1 to the cell pellet * Vortex, then let settle until most of the air bubbles have vanished 3. Encapsulation 1. Take up the cell solution with the syringe needle. NOTE: The solution is quite viscous, so care should be taken when aspirating in order to avoid any air bubbles 2. One drop into each eprouvette from a height of about 10 cm – capsule formation should occur instantly and the capsule sink to the ground. NOTE: Avoid air bubbles! 3. After 10 min of hardening time, remove the capsules from the hardening bath with a spatula and transfer them into a prepared glass vial (see step 1B) and put the vial on a 360° shaker (approximately 50 rpm) 4. Draw samples of 100 µL supernatant and one capsule of each ampoule at every timepoint * 0 h; 0.5 h; 1 h; 2 h and 24 h * Plate (capsules were ruptured before plating using a sterile toothpick) * Incubate at 37 °C for 24 h Expected results: Only plates of capsules should be overgrown. This shows that cells survive within the capsule but did not escape into the supernatant. Thiolation Altered microwave method ([1](https://doi.org/10.1021/acsami.5b06078))([2](https://doi.org/10.3390/pharmaceutics13060854))([3](https://doi.org/10.1016/j.ijpharm.2020.119040)) Reagents * 1 g chitosan, low molecular weight (Sigma Aldrich) * 1.4 g thiourea (Sigma Aldrich) * 50 mL 1 % acetic acid * 1.6 mL 37 % HCl * 1 M NaOH * Aceton Method * Solve 1 g chitosan (low viscosity) in 50 mL 1 % acetic acid in a 100 mL round bottom flask with a magnetic stirrer * Weigh 1.4 g thiourea under the hood with safety measures for toxic substances + Add 1.6 mL 37 % HCl + Add 2 mL distilled water + Add about 0.5 mL distilled water to wash the remaining thiourea out * Microwave + 250 W for 45 minutes in the microwave (no automatic temperature regulation). The temperature should be around 90 °C, if it is higher manually regulate it down. + 1 M NaOH for hydrolyzation, the clear solution turns cloudy (pH = 8-9) + 250 W for 5 minutes with no temperature regulation * Let it cool down to room temperature and neutralize it with HCl * Precipitate the solution in acetone * Filtrate and wash it three times with acetone * Dry freeze the product References (1) Chauhan, K., Singh, P., & Singhal, R. K. (2015). New Chitosan-Thiomer: An Efficient Colorimetric Sensor and Effective Sorbent for Mercury at Ultralow Concentration. ACS applied materials & interfaces, 7(47), 26069–26078. <https://doi.org/10.1021/acsami.5b06078> (2) Grosso, Roberto & De Paz, M. Violante. (2021). Thiolated-Polymer-Based Nanoparticles as an Avant-Garde Approach for Anticancer Therapies—Reviewing Thiomers from Chitosan and Hyaluronic Acid. Pharmaceutics, 13, 854. <https://doi.org/10.3390/pharmaceutics13060854> (3) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. <https://doi.org/10.1016/j.ijpharm.2020.119040> Synthesis of Cellulose Sulfate ProtocolMaterials * Chemicals: + Dry DMA (in molecular sieve) + Water free LiCl + SSP (spruce sulfite pulp from MODO PAPER) + NaOH (1 M) + SO3/Py + Acetone + 500ml Ethanol (96 % but 100 % would be best) * Dialysis membrane and clips * Syringe * Ultrasound bath * 2-neck-piston with septum * Cooler * Magnetic stirrer with implemented heater * Vacuum pump Preparation of SSP/DMA pulp * 10 g of SSP mashed with 1 L distilled water to a fine pulp * Vacuum filtrate the water out * Wash carefully with 500 mL ethanol and filter it (solvent exchange) * Wash and filter it 2-3 x with DMA * Fill the pulp in a glass bottle and add about 450-500 mL DMA * Place it overnight on a shaker * Filtrate the pulp and DMA mixture * Wash 2 x with DMA again and press as much as possible the solution out * Weigh the pulp and store it in a fridge * Warm the pulp to room temperature before use * Preparation of DMA/LiCl (9%) * 13.5 g LiCl + 150 mL DMA * Stirring at 60°C until completely dissolved (approx. 1 h) * Cool down to room temperature Preparation of SO3/Py in DMA * 6 g SO3/Py + 50 mL DMA * 15-20 min in ultrasound bath at 40 °C (until completely dissolved) * Cool down to room temperature Synthesis of Cellulose-Sulfate Protocol ([1](https://doi.org/10.1016/j.eurpolymj.2019.07.030)) * Dissolve 1.5 g cellulose in 150 mL DMA/LiCl (9 %) * Stir for 12 h at room temperature * Add 6 g SO3/Py in 50 mL DMA at 80 °C + 10 x 5 mL over 2 h * Stir for 1h at 80 °C * Cool down to room temperature * Precipitate in 750-1000 mL of acetone for 1 h * Wash 3 x with acetone and filtrate + Solution must be clear, otherwise filtrate again * Neutralize after 1 h by dissolving in 75 mL of NaOH * Dialyze after 1 h Fill the solution in dialysis membranes and dialyze it against distilled water+ Fill a big container with distilled water and add the full dialysis membranes + Put it in a sink and slightly open the distilled water so that a little amount of water drops in * Freeze dry when pH = 7 References Muhitdinov, B., Heinze, T., Turaev, A., Koschella, A., & Normakhamatov, N., (2019). Homogenous synthesis of sodium cellulose sulfates with regulable low and high degree of substitutions with SO3/Py in N,N-dimethylacetamide/LiCl. European Polymer Journal, 119, 181-188. <https://doi.org/10.1016/j.eurpolymj.2019.07.030> Cationic, thiolated polyDADMAC-chitosan solution (own solution) ProtocolMaterials * Thiolated chitosan * PolyDADMAC high * 0.9 % NaCl * 0.1 M HCl Method * Dilute PolyDADMAC high with 0,9 % NaCl to 5 % * Dilute thiolated chitosan with 0.1 M HCl to 1.6 % * Mix the 2 solutions in a 1:4 ratio (thiolated chitosan : polyDADMAC) SSP capsule in PolyDADMAC ProtocolMaterials * Freeze dried cellulose sulfate * PolyDADMAC (high molecular weight, 40 %) * Tween 80 * 0.9 % NaCl * Distilled water * Syringe Method Capsule solution (polyanion): * Dissolve 0.06 g of cellulose sulfate in 1 mL distilled water (= 6 %) * Vortex Solution (polycation): * Dilute 4 mL PolyDADMAC in 32 mL NaCl (0.9 %) * Add a small drop of Tween 80 Drop the polyanion solution with a syringe from a height of 10-15 cm in the polycation solution. Disulfide-bridge-test Protocol Quantification of disulfide substructures ([1](https://doi.org/10.1016/j.ijpharm.2020.119040)) Solution preparation * TRIS-buffer: + 0.788 g TRIS-HCl in 100 mL distilled water OR + 121.14 g/mol Trizma-base: 0.606 g/100 mL distilled water + Adjust the pH to 7.6 with HCl * Fresh NaBH4 solution (4%): + 600 mg + 15 mL distilled water * Phosphate buffer 1 M pH = 8.0: + 0.95 g KH2PO4 + 15.408 g Na2HPO4 \*12 H2O in 50 mL distilled water + Adjust pH to 8 if necessary * Fresh Ellman’s reagent: + 12 mg (5,5’-Di-thio-bis-(2-Nitro-benzoic Acid) in 3 mL Ellman’s buffer Sample preparation * Weighing 3 x 0.5 mg polymer in 15 mL falcon tubes * add to each 500 µL TRIS buffer and let it soak for 30 minutes + Chitosan: let it soak in 350 µL distilled water and after 30 minutes add 150 µL TRIS buffer Calibration * 1 mg L-cysteine-HCl in 1 mL TRIS buffer * Dilution series: (A = 50 µg) 1. 100 µL stock solution + 900 µL TRIS buffer (1:10) 2. 500 µL solution A + 500 µL TRIS buffer (1:2) 3. 500 µL solution B + 500 µL TRIS buffer (1:2) 4. 500 µL solution C + 500 µL TRIS buffer (1:2) 5. 500 µL solution D + 500 µL TRIS buffer (1:2) 6. 500 µL solution E + 500 µL TRIS buffer (1:2) + throw 500 µL away 7. 500 µL TRIS buffer (blank) Test 1. Add to each sample and calibration solution 1 mL fresh NaBH4 solution 2. Incubate the samples at 37 °C for 1 h at a shaker water bath with slightly open lids (!) 3. + 250 µL 5 M HCl (cautiously) to stop the reaction 4. +1 mL 1 M phosphate buffer to neutralize it 5. + 250 µL fresh Ellman’s reagent 6. Incubate it for 90-120 min in the dark at room temperature 7. Centrifuge the sample for 5 min at 500 rpm if the solution is not clear 8. Measure 2 x 100 µL of the samples in a microtiter plate (absorbance: 450 nm, optimize gain) References (1) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. <https://doi.org/10.1016/j.ijpharm.2020.119040> Ellman’s Test Protocol Test for thiolgroups incl. Cystein ([1](https://doi.org/10.1016/j.ijpharm.2020.119040)) Solution preparation * Ellman’s buffer (Phosphatbuffer 0.5 M pH = 8.0): + 0.95 g KH2PO4 + 15.408 g Na2HPO4\*12 H2O in 100ml distilled water + Adjust pH to 8 * Fresh Ellman’s reagent + 3 mg (5,5-di-thio-bis-(nitro-benzoic acid) in 10ml Ellman’s buffer Sample preparation * Weighting 3x 0,5-1,0 mg polymer in 1,5 ml Eppendorf tubes * add to each 500 µl Ellman’s buffer and let it soak for 30 minutes Calibration * 1mg L-Cystein-HCl in 1ml Ellman’s buffer = stock solution (Cystein = 121.16 g/mol) * Dilution series: (A = 25 µg) 1. 50 µl stock solution + 950 µl Ellmans buffer (1:20) 2. 500 µl solution A + 500 µl Ellmans buffer (1:2) 3. 500 µl solution B + 500 µl Ellmans buffer (1:2) 4. 500 µl solution C + 500 µl Ellmans buffer (1:2) 5. 500 µl solution D + 500 µl Ellmans buffer (1:2) 6. 500 µl solution E + 500 µl Ellmans buffer (1:2) + throw 500 µl away 7. 500 µl Ellmans’s buffer (blank) Test 1. Add to each sample and calibration solution 500 µl fresh Ellman’s reagent 2. Incubate it for 90-120 min in the dark at room temperature 3. Centrifuge the sample for 5 min at 500 rpm 4. Measure 2x100 µl of the samples in a microtiter plate (absorbance: 450 nm, optimize gain) References (1) Hussain Asim, M., Nazir, I., Jalil, A., Matuszczak, B., & Bernkop-Schnürch, A. (2020). Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion. International journal of pharmaceutics, 577, 119040. <https://doi.org/10.1016/j.ijpharm.2020.119040> Tensile studies Protocol: Mucoadhesion test ([1](https://doi.org/10.1016/s0378-5173%2899%2900387-7)) Materials: * Porcine intestine (room temperature) - 90-200 mg thiolated chitosan - 90-200 mg unmodified chitosan - (1.5-3.0 mL PBS buffer [0.1 M and pH = 6,8]) - Textur-Analysator TA.XTplus (Stable Micro Systems) Measure parameters * Applied force: 0.100 N * Return distance: 20.00 mm * Contact time: 90 sec * Release force: 0.029 N Method * 30 mg of thiolated chitosan and unmodified chitosan pressed into 5.0 mm diameter flat-faced discs (triplicate or 2x triplicates) ![](https://static.igem.org/mediawiki/2021/7/74/T--BOKU-Vienna--Experiments_Scaffolds_01.jpeg) Pressed discs * Cut about 2 cm of the intestine off and slice it carefully open ![](https://static.igem.org/mediawiki/2021/0/0a/T--BOKU-Vienna--Experiments_Scaffolds_03.jpeg) Spread out intestine * Carefully spread the intestine open and place it with the mucosa above on the plate without damaging it ![](https://static.igem.org/mediawiki/2021/e/e8/T--BOKU-Vienna--Experiments_Scaffolds_02.jpeg) Texture analyzer plate * Close it and eventually add 200 µl PBS buffer to simulate the intestine environment * Attach the test discs with double sided tape (there should be no tape sticking out) * Rotate the plate for each test so that the undamaged part of the mucosa is used for the measuring (about 4 tests for each prepared intestine slice) References (1) Bernkop-Schnürch, A., and Steininger, S. (2000). Synthesis and Characterisation of Mucoadhesive Thiolated Polymers. International Journal of Pharmaceutics 194.2, 239-47.[https://doi.org/10.1016/s0378-5173(99)00387-7](https://doi.org/10.1016/s0378-5173%2899%2900387-7) Rheological evaluation Viscosity test ([1](https://doi.org/10.1016/j.actbio.2021.08.016)) Materials: * 12 x 0.5 g porcine mucosa in 60 mm petri dish (preparation and purification are also described in the paper) * 200 mg thiolated chitosan * PBS buffer (50 mM, pH = 6.8) * Plate-plate rheometer (Thermo Scientific™ HAAKE MARS™, Thermo Fisher Scientific, Karlsruhe, Germany; rotor PP35Ti, D = 35 mm, gap 0.5 mm) ![](https://static.igem.org/mediawiki/2021/7/77/T--BOKU-Vienna--Experiments_Scaffolds_04.jpeg) Plate-plate rheometer Measure time * 0 h (triplicate) * 1 h (triplicate) * 2 h (triplicate) * 3 h (triplicate) Method * Incubate the mucosa at 37 °C for 30 minutes * Dissolve/suspend 200 mg thiolated chitosan in 10 mL PBS buffer 2 %(w/v) * Add 0.5 mL of the thiolated chitosan solution to the 3 petri dishes of 3 h, mix it and incubate it * After 1 h: add 0.5 mL of the thiolated chitosan solution to the 3 petri dishes of 2 h, mix it and incubate it * After another 1 h: add 0.5 mL of the thiolated chitosan solution to the 3 petri dish of 1 h, mix it and incubate it * After another 1 h (in total after 3 h since the first sample): add 0.5 mL of the thiolated chitosan solution to the one petri dish stir it and measure it quickly. * Clean the rheometer and repeat it with the other two sample of the measure time t = 0 h * Measure the other samples, gently scrape it off from the petri dish Measure parameters * Measured using an oscillatory strain sweep method with frequency = 1 Hz and temperature = 37 °C * Shear stress in a range of 0.5-500 Pa. * Samples equilibrated on the lower plate for 60 seconds after application until the start of the measurement References (1) Knoll, P., Le, N. N., Wibel, R., Baus, R. A., Kali, G., Asim, M. H., & Bernkop-Schnürch, A. (2021). Thiolated pectins: In vitro and ex vivo evaluation of three generations of thiomers. Acta biomaterialia, S1742-7061(21)00543-2. Advance online publication. <https://doi.org/10.1016/j.actbio.2021.08.016> Chitosan and thiolated chitosan HCl Protocol For water soluble chitosan hydrochloride for cytotoxicity test ([1](https://doi.org/10.1007/s002890050448)) Materials: * 1 g Chitosan or thiolated chitosan * 50 mL 0.1 M HCl * Dialysis membrane * Dialysis clips Method: * Solve chitosan or thiolated chitosan in 50 mL 0.1 M HCl * Filter the non-soluble substances * Fill the solution in dialysis membranes and dialyze it against distilled water + Fill a big container with distilled water and add the full dialysis membranes + Put it in a sink and slightly open the distilled water faucet so that a little amount of water drops constantly in (or change the water regularly) * Check the pH at the next day if it is neutralized * If yes, freeze dry it References (1) Signini, R., Campana Filho, S. (1999). On the preparation and characterization of chitosan hydrochloride. Polymer Bulletin 42, 159–166. <https://doi.org/10.1007/s002890050448>

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# Results

To achieve our goal of site-directed delivery of fructan-degrading enzymes over a prolonged timespan the selected approach was three-fold.

* Firstly, the Cloning team aimed to engineer *L.plantarum* using Golden Gate Assembly to express the enzymes endo-inulinase,
  levanase, and invertase for the degradation of fructans to glucose.
* Secondly, the Scaffolds team intended to create a biocompatible, mucoadhesive scaffold from cellulose sulfate and Polydiallyldimethylammonium chloride (polyDADMAC) to encapsulate the transgenic bacteria and thus protecting it from extreme pH values and digestive enzymes.
* Lastly, the Cell Culture team would test the cytotoxicity of the created capsules in vitro using Caco-2 cells.

On this page, we present the achievements of our three teams through the course of our laboratory work from July until October.
Details, on how these approaches were performed can be found in the [Experiments and Protocol section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments) as well as in our chronologically
documented [laboratory journals](https://2021.igem.org/Team%3ABOKU-Vienna/Notebook).

Cloning

Cell culture

Scaffolds

# Cloning

To lay the groundwork for all Golden Gate Assembly steps, the sequences for the raw backbone 1 (=BB1), backbone 2 (=BB2), and backbone 3 (=BB3) with their
specific fusion sites (=FS) were planned and cloned in silico via Benchling. The BB1 and BB2 vectors with their respective fusion sites were kindly obtained
by our supervisors at BOKU Vienna, but for BB3 a novel vector was created by our Cloning team, using the pSIP403 vector as a basis. The pSIP403 vector however
contains five illegal sequences forbidden for the Type II-S system. To circumvent this, the plasmid was split into five parts and five forward and five reverse
primers for each part were designed. Using these primers, five DNA constructs (c1b, c2b, c3b, c4b and c5a) deficient of any illegal sites were amplified, which
were then used to assemble a novel BB3 via Golden Gate Assembly.

![](https://static.igem.org/mediawiki/2021/8/89/T--BOKU-Vienna--Results_Cloning_01.jpeg)

Plasmid of assembled raw BB3

The sequencing results of the five constructs used to assemble the raw BB3 showed a few mutations upon alignment with the in silico cloned vector.
However, these turned out to be of no significance as they did not impair the function of vital elements of the backbone.

![](https://static.igem.org/mediawiki/2021/4/4a/T--BOKU-Vienna--Results_Cloning_02.jpeg)

Raw BB3 sequencing result

With the novel BB3 as a basis, four different versions containing different fusion sites for more flexibility in cloning were created,
namely version A-B, A-C, A-D, and A-E. This allows to insert up to five constructs into BB3.

To achieve this, our supervisors at BOKU kindly provided us with BB2 containing the aforementioned fusion sites as inserts, which were then used to assemble our four
BB3 versions via Golden Gate Assembly. All four versions were first cloned in silico via Benchling, which were then used as templates to align our sequencing results
to ultimately confirm that our assembly of all BB3 vector variations was correct. As mentioned above, the shown point mutations did not result in any disfunction of the vector and can be attributed to mutations that naturally occurred over time.

❮

![](https://static.igem.org/mediawiki/2021/1/1d/T--BOKU-Vienna--Results_Cloning_03.jpeg)

![](https://static.igem.org/mediawiki/2021/9/97/T--BOKU-Vienna--Results_Cloning_04.jpeg)

![](https://static.igem.org/mediawiki/2021/e/e5/T--BOKU-Vienna--Results_Cloning_05.jpeg)

![](https://static.igem.org/mediawiki/2021/9/9d/T--BOKU-Vienna--Results_Cloning_06.jpeg)

❯

Empty BB3 with fusion sites AB, AC, AD, and AE

The first assembly step was to clone our promotors (p1 – p8), enzymes (endo-inulinase = Endi, invertase = Invi, levanase = Levi) with their respective secretion
signal A and B, and the terminators (ttCAT and pepNTT) into their corresponding BB1. The promotor p8 (=pGAP) in BB1 was already provided by our supervisors.
To align our sequencing results, first, every BB1 with the respective gBlock was cloned in silico via Benchling. All promoters were cloned into BB1 with fusion
site 1-2, all enzymes into BB1 fusion site 2-3, and all terminators into BB1 fusion site 3-4.

Below, see the sequencing results for all BB1 are depicted, each with the in silico cloned template on top and with the corresponding sequencing result on the bottom,
proofing the correctness of each BB1 construct.

❮

![](https://static.igem.org/mediawiki/2021/c/cb/T--BOKU-Vienna--Results_Cloning_07.jpeg)

![](https://static.igem.org/mediawiki/2021/e/eb/T--BOKU-Vienna--Results_Cloning_08.jpeg)

![](https://static.igem.org/mediawiki/2021/1/17/T--BOKU-Vienna--Results_Cloning_09.jpeg)

![](https://static.igem.org/mediawiki/2021/e/e6/T--BOKU-Vienna--Results_Cloning_10.jpeg)

![](https://static.igem.org/mediawiki/2021/c/c2/T--BOKU-Vienna--Results_Cloning_11.jpeg)

![](https://static.igem.org/mediawiki/2021/9/9e/T--BOKU-Vienna--Results_Cloning_12.jpeg)

![](https://static.igem.org/mediawiki/2021/0/0d/T--BOKU-Vienna--Results_Cloning_13.jpeg)

❯

Promotors in BB1

❮

![](https://static.igem.org/mediawiki/2021/2/2d/T--BOKU-Vienna--Results_Cloning_14.jpeg)

![](https://static.igem.org/mediawiki/2021/7/72/T--BOKU-Vienna--Results_Cloning_15.jpeg)

![](https://static.igem.org/mediawiki/2021/9/9d/T--BOKU-Vienna--Results_Cloning_16.jpeg)

![](https://static.igem.org/mediawiki/2021/d/d3/T--BOKU-Vienna--Results_Cloning_17.jpeg)

![](https://static.igem.org/mediawiki/2021/5/53/T--BOKU-Vienna--Results_Cloning_18.jpeg)

![](https://static.igem.org/mediawiki/2021/9/91/T--BOKU-Vienna--Results_Cloning_19.jpeg)

![](https://static.igem.org/mediawiki/2021/d/d4/T--BOKU-Vienna--Results_Cloning_20.jpeg)

❯

Enzymes in BB1

❮

![](https://static.igem.org/mediawiki/2021/2/23/T--BOKU-Vienna--Results_Cloning_21.jpeg)

![](https://static.igem.org/mediawiki/2021/4/43/T--BOKU-Vienna--Results_Cloning_22.jpeg)

❯

Terminators in BB1

The red fluorescent protein mCherry was also cloned into BB1 to be used as a reporter gene to quantify our promotors. This is discussed further below.

![](https://static.igem.org/mediawiki/2021/1/17/T--BOKU-Vienna--Results_Cloning_23.jpeg)

Having all promotors, enzymes, and terminators successfully cloned into BB1, the second step was to assemble them into BB2, creating a number of different
expression cassettes with varying promotor-enzyme-terminator-combinations. Through sequencing, it became apparent that clones suspected to be positive all either
had frameshift mutations, deletions, insertions, or even the whole enzyme was missing (examples see below), rendering most BB2 expression cassettes unusable for
further cloning steps

For instance, in the picture below the sequencing result for a BB2 expression cassette (BB2\_FSBC\_p3\_Levi\_pepNTT) can be seen.
Here, with clones 6.1 and 6.2 a shared deletion of the first base of the secretion signal can be observed. Clones 5.1 and 5.2 have a shared mutation in the promotor,
that results in a translational change (TAT to CTA results in S à \*).

![](https://static.igem.org/mediawiki/2021/8/8d/T--BOKU-Vienna--Results_Cloning_24.jpeg)

As our initially approach seemed to be too overwhelming for the *E.coli* to express correctly, we reconsidered our original approach and changed course.

First, we used the weaker promotors to try and circumvent overwhelming the *E.coli* with our strong promotors as we suspected that the metabolic burden was
too high for *E.coli*. However, this approach still did not result in any positive clones.

![](https://static.igem.org/mediawiki/2021/e/ed/T--BOKU-Vienna--Results_Cloning_25.jpeg)

Next, we attempted to simply amplify the PCR products after the second Golden Gate Assembly step into BB2, therefore making our way from BB2 to BB3 without transformation in *E.coli*. This however proofed to be ill-fated, as our amplified PCR-products were either of too little concentration for further assembly steps or they were unusable after too many amplification-rounds. Below an example of this can be seen on a preparative 1 % Agarose gel: The bands were smeared and not possible to cut out, therefore they could not be purified for further use.

![](https://static.igem.org/mediawiki/2021/2/28/T--BOKU-Vienna--Results_Cloning_26.jpeg)

As we were unable to properly clone our intended promotor-enzyme-terminator-combinations into BB3, we made the decision, on one hand, to try another promotor
to prove that our enzymes can be correctly expressed, and on the other hand by choosing a simpler protein to be expressed to proof our promotors were functioning.
For the latter, we chose the red fluorescent protein mCherry. The results for this promotor quantification can be seen in our
wiki page [Contributions](https://2021.igem.org/Team%3ABOKU-Vienna/Contribution).

To get to the bottom of the issue regarding our incorrectly expressed enzymes, we proceeded to clone our enzymes together with another promotor,
namely the inducible orfXP promotor. With this promotor, we were able to clone our three enzymes into BB3 correctly into *E.coli*.
However, as to not encounter any limitation of a too high metabolic burden with our initial approach of cloning all three enzymes into one expression
cassettes in BB3, we instead cloned each enzyme separately into BB3. As a next step, the three enzymes could be cloned into one expression cassette in
order to observe the effects of the metabolic burden on *Lactobacillus*. Due to time constraints, we will not conduct these experiments.

With this approach, our enzymes Endi KM A, Invi A, and Invi B with the terminator ttCAT and Levi with the terminator pepNTT were cloned correctly into BB3.
Furthermore, we were also able to transform the expression cassettes containing these constructs into *L.plantarum*, as seen in the sequencing results below.

![](https://static.igem.org/mediawiki/2021/7/77/T--BOKU-Vienna--Results_Cloning_27.jpeg)

❮

![](https://static.igem.org/mediawiki/2021/c/c8/T--BOKU-Vienna--Results_Cloning_28.jpeg)

![](https://static.igem.org/mediawiki/2021/3/32/T--BOKU-Vienna--Results_Cloning_29.jpeg)

![](https://static.igem.org/mediawiki/2021/1/12/T--BOKU-Vienna--Results_Cloning_30.jpeg)

![](https://static.igem.org/mediawiki/2021/1/14/T--BOKU-Vienna--Results_Cloning_31.jpeg)

❯

BB2 transformed in *E.coli*

❮

![](https://static.igem.org/mediawiki/2021/8/86/T--BOKU-Vienna--Results_Cloning_32.jpeg)

![](https://static.igem.org/mediawiki/2021/2/28/T--BOKU-Vienna--Results_Cloning_33.jpeg)

![](https://static.igem.org/mediawiki/2021/d/d9/T--BOKU-Vienna--Results_Cloning_34.jpeg)

![](https://static.igem.org/mediawiki/2021/3/32/T--BOKU-Vienna--Results_Cloning_35.jpeg)

❯

BB3 transformed in *E.coli*

❮

![](https://static.igem.org/mediawiki/2021/5/56/T--BOKU-Vienna--Results_Cloning_36.jpeg)

![](https://static.igem.org/mediawiki/2021/9/99/T--BOKU-Vienna--Results_Cloning_37.jpeg)

![](https://static.igem.org/mediawiki/2021/f/f1/T--BOKU-Vienna--Results_Cloning_38.jpeg)

![](https://static.igem.org/mediawiki/2021/f/f6/T--BOKU-Vienna--Results_Cloning_39.jpeg)

❯

BB3 transformed in *L.plantarum*

However, we were not able to obtain promising clones of Endi KM B in BB3. Nevertheless, we could identify promising *E.coli* clones carrying the expression cassette of the inducible promoter with the enzymes Endi AF A and Endi AF B. To this date, these clones still need to be sequenced and transformed into *L.plantarum*.

The next step with our transformed *L.plantarum* was to prove that the enzymes are indeed expressed and secreted after induction.

 We aimed to prove the secretion of our enzymes by performing a His-tag purification of the supernatant and afterwards perform an SDS-PAGE stained with Coomassie blue to detect the proteins. Unfortunately, we could not detect any proteins with this method. Therefore, we aimed for a more sensitive method. This allows us to analyse the supernatant of previously induced liquid cultures of the clones in a straightforward manner. Subsequently, the membrane was stained using an Anti-His HRP conjugate antibody. This provided us an easy and quick proof that the enzymes are secreted into the supernatant.

![](https://static.igem.org/mediawiki/2021/1/18/T--BOKU-Vienna--Results_Cloning_40.jpeg)

As seen in the picture, we achieved to detect His-tagged proteins in almost all supernatants of our clones. Differences in strength of the signal are most likely due to concentration differences as a result of different amounts of applied samples as the membrane unfortunaletly clogged while loading. We also analysed the His-tagged purified samples and could not detect any protein there. Since we could detect the enzymes in the supernatant and additionally also the positive controls shows a signal, we assume that the protein concentration after purification was too low to be detected.

# Discussion

At the time of the Wiki-freeze, we achieved to clone the enzymes endo-inulinase with secretion signal A and invertase with secretion signal A&B with ttCAT as terminator as well as the enzyme levanase with terminator pepNTT with the inducible promotor orfXP into one Backbone 3 with the fusion site AB. Additionally, we successfully transformed each construct into *L.plantarum* and showed that our enzymes are secreted into the supernatant. In a final step we aim to prove that our enzymes are able to degrade fructans, but this remains subject of future experiments.

The Caco-2 cells were thawed, and the media was regularly changed according to protocol.

The cells were made sure to be at 80 – 90 % confluency before passaging them.

Caco-2 cells, 2 days after the 3rd passage:

![](https://static.igem.org/mediawiki/2021/e/ed/T--BOKU-Vienna--Results_Cell-Culture_01.jpeg)

Caco-2 cells at 90% confluency:

![](https://static.igem.org/mediawiki/2021/c/c3/T--BOKU-Vienna--Results_Cell-Culture_02.jpeg)

We had our Caco-2 cells in active culture for six passages. Unfortunately, due to limited time in our Cell Culture Lab and the slower than anticipated progress
in our Cloning and Scaffold labs, we were unable to finally test the cytotoxicity of our capsules.

However, a protocol of how we would have undergone the Resazurin assay for testing the toxicity of the created capsules in vitro using Caco-2 cells can be found
in the [“Protocols” section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments).

# Capsule synthesis

At the beginning we focused on synthesizing our own cellulose sulfate to use as capsules. There were two options that we found promising: the sulfation
of cellulose in ionic liquids or in Dimethylamine (DMA)/LiCl, both using SO3/Py as sulfating reagent. For the first experiments with DMA/LiCl microcrystalline cellulose was
used but because of its light molecular weight, it did not form capsules when dripping it into the PolyDADMAC solution. Thus, cellulose of higher molecular
weight was needed. Our candidates were spruce sulfite pulp (SSP) and cotton linters (CL), they were used for the following synthesis. After analyzing the samples,
it was shown that the SSP-synthesis provided the best results for building capsules, and the sulfating rate was the highest using DMA/LiCl instead of the ionic
liquid because of its lower viscosity. The respective protocol can be found in the ["Protocol" section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments).

The best result was provided with 4 mol equivalents of SO3/Py, which you can not only see in the capsule building process but also in the FTIR spectrum.

![](https://static.igem.org/mediawiki/2021/4/43/T--BOKU-Vienna--Results_Scaffolds_01.jpeg)

Our synthesized cellulose sulfate (“SSP\_IV”, in dark blue) was compared to native cellulose (“Reference Avicel PH-101", in orange. The peak at 811.6cm-1 shows
C-O-S bonds and the signal at 1217cm-1 is characteristic for S=O bonds, both are clear evidence for sulfate groups.

Furthermore, the bath solution had also a significant influence on the capsules. In high molecular PolyDADMAC the capsules were more stable than in medium molecular
PolyDADMAC. Also, a detergent (Tween 80) had to be added to lower the surface tension of PolyDADMAC so that the capsules could be built more easily.
(In experiments with *E.coli* we noticed that Tween 80 was too aggressive for living cells.)

The best outcomes of sulfated SSP capsules are listed in the tables below:

| Sample name | SO3/Py equivalent | PolyDADMAC high 4% (diluted in 0.9% NaCl) |
| --- | --- | --- |
| SSP IV | 3 | + |
| SSP 16.2 | 6 | + |
| SSP VI | 4 | ~ |

In the first table, you can see 3 different samples in the same solution. Although our best sample (SSP VI, 4mol equivalents) was not very stable compared to
the other capsules in PolyDADMAC high 4% (diluted in 0.9% NaCl), it showed better results in other bath solutions. You can see the effect of the bath solution
on the capsule stability in the second table.

| Sample name | SO3/Py equivalent | PolyDADMAC high 4% (diluted in 0.9% NaCl), Tween 80, thiolated chitosan | PolyDADMAC high 4% (diluted in destilled water), Tween 80, thiolated Chitosan | Chitosan, 0.9% NaCl 1:4 | PolyDADMAC high 4% (diluted in 0.9% NaCl) | PolyDADMAC high 3% (diluted in 0.9% NaCl), chitosan |
| --- | --- | --- | --- | --- | --- | --- |
| SSP VI | 4 | + | ~ | - | ~ | + |

See also SSP capsule in PolyDADMAC Protocol in the ["Protocol" section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments)

In our journey to synthesize scaffold forming cellulose sulfate, we were confronted with many challenges. More details are described in the [engineering cycle](https://2021.igem.org/Team%3ABOKU-Vienna/Engineering).

 We achieved the best results with spruce sulfit pulp and 4 mol equivalents of SO3/Py. To test their stability, we put the empty scaffolds in distilled water and shook them over several days. They were still stable, but due to time constraints further tests and steps for optimization could not be conducted. Instead, we used the cellulose sulfate solution of the Cell-in-a-box kit from Austria Nova for the pore size test, which our system is based of.

# Pore size & Cell escape

As a key part of our Friendzyme delivery platform, we chose to encapsulate our living cells into a scaffold functioning as a biocontainer. The main requirement to the scaffold itself is thus, that the bacteria remain inside while the enzymes and nutrients are able to freely diffuse in and out. The only way to achieve this is by finding an optimal pore size that allows both of these properties.
We decided to demonstrate the functionality by assessing both the retention of cells (“Cell escape assay”) and the diffusion of small molecules (“Enzyme diffusion assay”).
At the time we started the tests, we did not have our enzyme expressing lactobacilli ready. However, we need cells with an antibiotic resistance because some of our components were not sterile. Thus we decided to encapsulate some of our *E.coli* cells instead. They are slightly smaller in size than lactobacilli but if we were able to demonstrate that they were retained by the scaffold, automatically the same would be true for larger cells. (see ["Protocol" section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments)) The enzyme escape was tested separately to get better and clearer measurement results. (see ["Protocol" section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments)) For encapsulation, we used the Cell-in-a-Box kit from Austrianova as well as our own polyDADMAC solution with thiolated chitosan.

## Cell escape assay

In the cell escape assay, solution 1 (kit) was first mixed with E.coli as described in the protocol, then dripped into the diluted solution 2 (kit) to encapsulate the bacteria. The resulting capsules were then placed in a PBS buffer. At certain time points, samples of capsules and of the supernatant were taken and plated out. After incubating the plates for 24 hours we got clear results: no cells grew on the plates of the supernatant. This indicates that the pores of the scaffolds were small enough so that the *E.coli* could not escape.

❮

![](https://static.igem.org/mediawiki/2021/1/1f/T--BOKU-Vienna--Results_Scaffolds_Pore_01.jpeg)

![](https://static.igem.org/mediawiki/2021/7/7a/T--BOKU-Vienna--Results_Scaffolds_Pore_02.jpeg)

![](https://static.igem.org/mediawiki/2021/e/e0/T--BOKU-Vienna--Results_Scaffolds_Pore_03.jpeg)

❯

Supernatant after 0 hours, 3 hours, and 24 hours

We also performed tests to see if the scaffold would be harmful to the cells. For this, we mechanically broke down the scaffold, after down after 2 and 24h and plated out the insides. After incubation for 48 h at 37 °C, we saw that colonies were growing on the plates. Thus proving that the encapsulation was not harmful to the cells.

❮

![](https://static.igem.org/mediawiki/2021/e/e4/T--BOKU-Vienna--Results_Scaffolds_Pore_04.jpeg)

![](https://static.igem.org/mediawiki/2021/0/08/T--BOKU-Vienna--Results_Scaffolds_Pore_05.jpeg)

![](https://static.igem.org/mediawiki/2021/f/f9/T--BOKU-Vienna--Results_Scaffolds_Pore_06.jpeg)

❯

Scaffold after 0 hours, 3 hours, and 24 hours

The same test was performed with our own prepared solution (see ["Protocol" section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments)), where we mixed PolyDADMAC with thiolated Chitosan. Same as with the kit solution, no cells could be found in the supernatant. Most of the plates of the capsules were overgrown, but some of them were also empty. As soon as there was a little air bubble in the scaffold the solution could enter the scaffold. One reason for the instability could be the shape, which was more a drop instead of a sphere. Another reason is the molecular weight of solution 1, which did not match that well with our self-prepared solution.

## Enzyme diffusion essay

We tested the enzyme escape by measuring the absorbance at 280nm, the absorption maximum of proteins, with a NanoDrop spectrophotometer.
Instead of encapsulating enzymes we chose to try to encapsulate Bovine Serum Albumin (BSA) for cost and convenience reasons. BSA is larger than our enzymes, but if it was able to exit the scaffoldy our expressed enzymes would be as well.
At first, we mixed the protein with solution 1 (containing cellulose sulfate) and dropped it into the diluted solution 2 (containing polyDADMAC) to form a capsule. It was then placed in PBS buffer. To measure the absorbance, we took samples of the supernatant of the capsules after 0 min, 30 min, 60 min, 120 min and 24 h. This was done in triplicates.

![](https://static.igem.org/mediawiki/2021/4/45/T--BOKU-Vienna--Results_Scaffolds_Pore_07.jpeg)

![](https://static.igem.org/mediawiki/2021/3/30/T--BOKU-Vienna--Results_Scaffolds_Pore_08.jpeg)

We first created a calibration curve using BSA standards dissolved in PBS. When measuring our samples, the absorbance in the beginning was always very low but noticeably increased over time. We thus were able to show that proteins diffused out of the capsule. However, the results indicate quite clearly, that diffusion of BSA out of the capsule in the beginning occurs only very slowly. Almost all of the BSA is released after several hours of incubation. Thus, the timeframe over which diffusion occurs was too long for our envisioned application. This shows that further adjustments of the pore size will be necessary in the future.

We concluded that the pore size is small enough to keep the bacteria from escaping, but not large enough to allow all of the proteins to escape. It is possible that the diffusion of our enzymes might occur faster than that of the larger BSA, however this needs further investigation using enzymes of the same size as the ones we want to secrete.

## Summary

We proved that the pore size of our scaffold was small enough to keep the bacteria inside and for some protein to diffuse out. However, the flux out of the scaffold was not really satisfactory. Due to the time restraint, we unfortunately had no room for further experiments, so some more pore size fine-tuning would be necessary in the future and also our actual secreted proteins should need to be used in order to assess the actual efficiency of protein diffusion.

For the scaffold formed out of our own solution, additional experiments like the optimization of the concentration of the thiolated chitosan, are needed to achieve better results.

# Thiolation results

One of the remarkable and innovative characteristics of our scaffold is its mucoadhesive property. It sticks to the mucosal surface of the human intestine thus prolonging the time it can remain there and release its therapeutic enzymes. This is achieved using a method called thiolation, where thiol groups are added which form disulfide-bridges with cysteine residues of the mucosa.

![](https://static.igem.org/mediawiki/2021/a/a7/T--BOKU-Vienna--Results_Scaffolds_02.jpeg)

Thiolated cellulose sulfate Ellman’s test:

|  | Sample absorbance | Thiol concentration [mM] |
| --- | --- | --- |
| Average | 0.07578 | 0.01529411 |

![](https://static.igem.org/mediawiki/2021/a/a3/T--BOKU-Vienna--Results_Scaffolds_03.jpeg)

'Thiolated cellulose sulfate' disulfid-bridge test:

|  | Sample absorbance | Thiol concentration [mM] |
| --- | --- | --- |
| Average | 0.053213 | 0.013965 |

As we can see, the sample was slightly thiolated. Unfortunately, we noticed that the cellulose sulfate was desulfated, apparently due to of the microwave irradiation during thiolation. This rendered the cellulose water insoluble and the negative charge of the sulfate, which is needed for the forming of a polyelectrolyte complex, was lost. Even though the thiolation was successful, we could not use this cellulose.

![](https://static.igem.org/mediawiki/2021/3/34/T--BOKU-Vienna--Results_Scaffolds_04.jpeg)

Thiolated chitosan disulfid-bridge test:

|  | Sample absorbance | Thiol concentration [mM] |
| --- | --- | --- |
| Thio\_chitosan\_1 | 0.051 | 0 |
| Thio\_chitosan\_2 | 0.0494 | 0 |
| Thio\_chitosan\_3 | 0.1538 | 0.374766 |
| Thio\_chitosan\_4 | 0.0643 | 0.039686 |
| Thio\_chitosan\_5 | 0.05507 | 0.005129 |

![](https://static.igem.org/mediawiki/2021/0/0e/T--BOKU-Vienna--Results_Scaffolds_05.jpeg)

Thiolated chitosan Ellman's test:

|  | Sample absorbance | Thiol concentration [mM] |
| --- | --- | --- |
| Thio\_chitosan\_1 | 0.0984 | 0.063191 |
| Thio\_chitosan\_2 | 0.07187 | 0.026507 |
| Thio\_chitosan\_3 | 0.06957 | 0.023327 |
| Thio\_chitosan\_4 | 0.0706 | 0.247511 |
| Thio\_chitosan\_5 | 0.0657 | 0.017976 |

The most promising product was thio\_chitosan\_3 since disulfid-bridge test showed us, that thio\_chitosan\_3 had the highest concentration of disulfid-brides even as the Ellmann’s test showed us there were a a few thiol groups on every sample. There were more disulfide-bridges than thiol groups, which leads to the conclusion that intermolecular disulfid-bridges were formed during the thiolation of thio\_chitosan\_3. This of course, was highly undesired as we wanted our thiol groups to later engage with cysteine residues on the mucosa and not be already saturated by bonds with thiolated chitosan molecules.

## Texture analyzer - Tensile studies

The texture analyser allowed us to show whether our thiolated chitosan had mucoadhesive properties. The freeze dried product and native chitosan was compressed into flat-faced disks. The disks were pressed against the mucosa layer of porcine intestine with a specific force for 90 seconds to form disulfid-bridges. Then the detachment force was measured.

Native chitosan from crab shells:

| Triplicate | N | N\*sec |
| --- | --- | --- |
| 1 | 0.036 | 0.890 |
| 2 | 0.061 | 0.118 |
| 3 | 0.071 | 0.605 |
| Avg. | 0.056 | 0.538 |
| SD. | 0.018 | 0.390 |

Maximal detachment force (MDF)= 56.00 mN
Total work of adhesion (TWA) = 53.77 mJ

Native chitosan from crab shells:

| Triplicate | N | N\*sec |
| --- | --- | --- |
| 1 | 0.152 | 1.875 |
| 2 | 0.160 | 0.949 |
| 3 | 0.165 | 1.462 |
| Avg. | 0.159 | 1.429 |
| SD. | 0.007 | 0.464 |

Maximal detachment force (MDF)= 159.00 mN
Total work of adhesion (TWA) = 142.9 mJ

As the tests showed, the adhesion work and detachment force of the thiolated sample is much higher than the native chitosan. We can thus conclude that the thiolation increased the mucoadhesive property of the chitosan.

## Rheological evaluation

The rheometer tests the viscosity of our samples. We added suspended thiolated chitosan and native chitosan to porcine mucosa. They were incubated for 0 h and 3 h at 37 °C. When disulfide bridges are formed, the viscosity increases. This effect was indeed observed with our thiolated chitosan, thus showing that disulfide bridges are formed and mucoadhesivity is achieved.The respective protocol can be found in the ["Protocol" section](https://2021.igem.org/Team%3ABOKU-Vienna/Experiments).

Native chitosan from crab shells:

|  | 0 h (Pas) | 3 h (Pas) |
| --- | --- | --- |
| 1 | 22.40 | 27.41 |
| 2 | 21.03 | 64.75 |
| 3 | 12.68 | 53.38 |
| Avg. | 18.85 | 48.51 |
| SD. | 5.22 | 19.14 |

Modified chitosan:

|  | 0 h (Pas) | 3 h (Pas) |
| --- | --- | --- |
| 1 | 62.94 | 170.70 |
| 2 | 76.90 | 161.70 |
| 3 | 81.67 | 236.40 |
| Avg. | 73,84 | 189,60 |
| SD. | 9,73 | 40,78 |

As the tests showed, the viscosity of the thiolated sample increased over time and is much higher than the native chitosan. More disulfid-bridges were formed, this showed that the thiolation worked.

## NMR evaluation

![](https://static.igem.org/mediawiki/2021/c/cd/T--BOKU-Vienna--Results_Scaffolds_06.jpeg)

Since the thiolation of chitosan worked and through the solid-state NMR, we can see that there is no peak at exactly 180 ppm. A sharp peak there would indicate that the presence of thiourea residue and/or the intermediate product isothiouronium ion. Thus, we can see the synthesis of pure thiolated chitosan was successful.

![](https://static.igem.org/mediawiki/2021/3/37/T--BOKU-Vienna--igem_vienna_logo.svg)

### About us

We are a team of students from different Viennese universities enthusiastic about advances and innovation in synthetic biology.

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