iGEM 2023 — Team nu-kazakhstan

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# Notebook

## Notebook

---

All the lab work, including intermediate results, is added to the labbook. Since our project consisted of two main parts - hydrogel synthesis and protein expression - we were simultaneously working with hydrogel synthesis and plasmid construction. Unfortunately, due to several factors independent of us, we are only expecting to receive our ordered plasmid shortly. Therefore, below, only hydrogel-related experiments are listed.

Lab notebook

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* Content
* [Cycle 1](#cycle1)
* [Cycle 2](#cycle2)
* [Cycle 3](#cycle3)
* [Cycle 4](#cycle4)
* [Cycle 5](#cycle5)
* [Cycle 6](#cycle6)
* [References](#References)

# Engineering Success

## Cycle 1 cohort: Adjustment of the general plan

---

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/cycle.png)

**Initial Project Design:**
1. To create a systemic cancer treatment
* Administration route- intravenous injection
2. Hydrogel system: pH-sensitive swelling of the hydrogel
3. Tumor types: All (Generalized treatment)

**Learning and Optimization:**

We organized several meetings with our PIs, Professors, National Biotechnology Center, and other field experts to evaluate our initial project design. After getting feedback from our primary and secondary PIs (Prof. Ivan Vorobyev and Prof. Elina Mun), Professor Timo Burster, doctors from the National Scientific Center for Oncology and Transplantation, our mentors and conducting an excessive literature review, we were able to learn about the downsides of our initial plan. Afterwards, we optimized the plan and made a new design.

**Final Project Design:**

1. To create a local cancer treatment
* Administration route- intratumoral injection

2. Hydrogel system: Temperature-dependant solidification of the hydrogel, pH-dependant drug release, and specific adhesion to cancer cells.

3. Tumor types: Solid carcinomas

**Rationales behind the changes:**

1. Systemic treatment would be less efficient and practical compared to the local treatment. Moreover, it would be highly difficult to minimize bacterial therapy side effects. As a result, we decided to change the administration route from intravenous injection to direct injection into the tumor site.

2. Since we decided to change the administration route, incorporating the hydrogel's pH sensitivity became redundant. However, it was suggested to adjust the properties of the hydrogel so that it would solidify at approximately 37℃ (body temperature). Such property will ensure that the gel can be easily administered into the tumor site in the liquid state and will not escape from the site after the injection. Additionally, our new hydrogel formulation allows cancer-specific gel adhesion.

3. We decided to narrow the scope of our treatment to carcinomas since, compared to other tumor types, local administration of the drugs via intratumor injection can be readily applied to treat carcinomas.

## Cycle 2 Cohort: Adjustment of the hydrogel formulation

---

We call this cycle a cohort since it includes multiple other small cycles.

### 1st Cycle:

**Design (Initial formulation):**
Initially, we chose Chitosan/Glycerol mixture as our hydrogel formulation.

**Build and Test:**
Hydrogel was prepared with Chitosan and Glycerol. Different proportions of reagents were used for several samples, and sol-gel transition behavior was evaluated using tube inversion method

**Learning and Optimization:**

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/figure-1-results-of-hydrogel.gif)

**Figure 1.** Chitosan/Glycerol hydrogels after 20 min in the water bath at 37°C

None of the Chitosan/Glycerol hydrogels exhibited temperature sensitivity. Hence, we did not observe solidification of the gels: all formulations remained liquid upon temperature increase. Since we were aiming for in situ forming hydrogel formation, we decided to change the composition of the gel.

### 2nd Cycle:

**Improve (New formulation):**
After conducting an additional literature review, we changed the hydrogel formulation to Chitosan and β-glycerophosphate mixture.

**Build and Test:**

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/figure-2-chitosan-glycerophosphate-hydrogel-right-after-the-synthesis-2.gif)

**Figure 2.** Chitosan/β-glycerophosphate hydrogel right after the synthesis

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/figure-3-chitosan-glycerophosphate-hydrogel-after-10-min-in-the-water-bath-at-37-c.jpeg)

**Figure 3.** Chitosan/β-glycerophosphate hydrogel after 10 min in the water bath at 37°C

Hydrogel was synthesized with 60 mg of Chitosan and 300 mg β-glycerophosphate dissolved in 2.5 mL of DI water. Sol-gel transition behavior was evaluated via tube inversion method.

**Learning and Optimization:**
The newly synthesized hydrogel was temperature-sensitive since we observed solidification at ~36℃. However, according to the literature, the average pore size of CS/GP hydrogel is 56.61 ± 18.82 μm [1]. Therefore, the pore size was too large to encapsulate bacteria efficiently since E. coli cells are about 1.0-2.0 micrometres long [2]. As a result, we decided to add a cross-linking agent to reduce the pore size.

### 3rd Cycle:

**Improve (New formulation):**
After an excessive literature review, hyaluronic acid was chosen as a suitable cross-linking agent. Unfortunately, since the reagent was unavailable in the laboratory, its salt (sodium hyaluronate) was added instead.

**Build and Test:**
Hydrogel was synthesized with 60 mg of Chitosan, 300 mg of β-glycerophosphate, and 15 mg sodium hyaluronate dissolved in 2.5 mL of DI water. Sol-gel transition behavior was evaluated via the tube inversion method.

**Learning and Optimization:**

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/figure-4-sem-imaging-of-chitosan-glycerophosphate-sodium-hyaluronate-4-hydrogel.jpg)

**Figure 4.** SEM Imaging of Chitosan/β-glycerophosphate/Sodium Hyaluronate (4%) hydrogel

During SEM imaging, we observed that sodium hyaluronate reduced the pore size of the gel. The average pore size was equal to 27.822 µM. However, the reduction was not sufficient to achieve the desired pore diameter. Hence, hyaluronic acid was ordered for further synthesis.

### 4th Cycle:

**Improve (New formulation):**
After realizing that hyaluronic acid can not be substituted with sodium hyaluronate, we decided to synthesize hydrogel with hyaluronic acid.

**Build and Test:**
Hydrogel was prepared with Chitosan, β-glycerophosphate, and hyaluronic acid. Five samples with different concentrations of the acid were prepared. The pore size of 5 samples with different concentrations of hyaluronic acid (0.5%, 1%, 2%, 3%, and 4% w/w) was evaluated via SEM microscopy.

**Learning and Optimization:**

**Table 1.** Correlation of the pore sizes of CH/GP/HA hydrogels to the proportion of hyaluronic acid.

| CH proportion | GP proportion | HA proportion | Total DI Water | Average pore size |
| --- | --- | --- | --- | --- |
| 60 mg | 300 mg | 0 mg | 2.5 mL | 56.61 ± 18.82 μm |
| 60 mg | 300 mg | 1.875 mg (0.5% w/w) | 2.5 mL | 18.39 μm |
| 60 mg | 300 mg | 3.75 mg (1% w/w) | 2.5 mL | 11.54 μm |
| 60 mg | 300 mg | 7.5 mg (2% w/w) | 2.5 mL | 3.94 μm |
| 60 mg | 300 mg | 11.25 mg (3% w/w) | 2.5 mL | Not Observed |
| 60 mg | 300 mg | 15 mg (4% w/w) | 2.5 mL | Not Observed |

Variations in the concentration of hyaluronic acid led to a significant change in the gel morphology. The last two hydrogels (3% and 4%) had a morphology of a film rather than a hydrogel matrix, and no pores were observed. 0.5% and 1% HA hydrogels had pores that could allow free migration of the cells to the outside environment. The formulation that would be capable of encapsulating E. coli cells most efficiently while also allowing macromolecules to diffuse freely to the surroundings is chitosan + glycerophosphate + 2% hyaluronic acid (w/w) hydrogel.

**Final formulation:**
Chitosan/β-glycerophosphate/Sodium Hyaluronate (2%) hydrogel was chosen as the best-fit model. To further test the encapsulation efficiency, a bacterial release test was conducted. To evaluate the cytotoxicity of the gel on E. coli cells, an MTT Assay was performed.

## 3rd Cycle Cohort: Adjustment of the Bacterial Release Test protocol

---

**Initial Design:**
Initial design of the Bacterial Release Test can be found in the Lab Notebook page.

**Build and Test:**
We conducted Bacterial Release Test according to the initial protocol.

**Learning and Optimization:**
After conducting the first run of the Bacterial Release Test, we adjusted our protocol and improved our basic microbiological techniques. The following changes to the procedure were made:
1. The bacterial release was evaluated by measuring the OD 600 nm of the samples loaded to the 48-well plate using a Varioskan microplate reader. Initially, the procedure was conducted by measuring OD600 nm of 1 mL of each sample in a cuvette using a spectrophotometer.
2. The plate layout was changed:
![](https://static.igem.wiki/teams/4782/wiki/engineering-success/figure-5-24-well-plate-layout-initial-bacterial-release-test-protocol.png)

**Figure 5.** 24-well plate layout (Initial Bacterial Release Test protocol)

3. Measurements were conducted more frequently for smoother and more accurate graph plotting

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/figure-6-48-well-plate-layout-final-bacterial-release-test-protocol.png)

**Figure 6.** 48-well plate layout (Final Bacterial Release Test protocol)

**Final Design:**
The Final Design for the Bacterial Release Test can be found on the Experiments page.

## 4th Cycle Cohort: Adjustment of the MTT Assay protocol

---

**Initial Design:**
Initially, we used the MTT Assay protocol that was designed for mammalian cells specifically.

**Build and Test:**
Procedure that was conducted can be found in the Lab Notebook page.

**Learning and Optimization:**
The results did not meet our expectations since all MTT Assay results were negative. We realized that it is necessary to adapt the MTT Assay protocol for bacterial cells specifically. For instance, washing the pellet with PBS and media discarding were omitted since, as practice shows, this step leads to complete elimination of bacteria.

**Final Design:**
The Final Design of the MTT Assay can be found in the Experiments page.

## 5th Cycle Cohort: Adjustment of the plasmid design

---

**Initial formulation:**
Colicin E1 & Colicin E1 immunity protein expression in response to N-Acyl homoserine lactones and L-lactate presence in the environment, followed by GFP reporter and immediate cell lysis. ColE1 is a widely used chemotherapeutic agent as well as the native E.coli protein used by it to kill cells by creating pores in the membrane. [3]
1. E.coli synthesizes ColE1 protein + ColE1 immunity protein in a micro tumor environment (L-lactate presence) and within high population density (AHL).
2. L-lactate presence is regulated by lldRPD, a wild-type promoter present in E.coli and serves as a metabolic mechanism that activates secondary carbon source degradation (lactate utilization) without glucose. [4]
3. N-Acyl homoserine lactones (AHL) is a quorum-sensing molecule that signals bacteria about the density of population. [5]
4. AND gate is used to express genes of interest only if those two conditions are satisfied: L-lactate presence and high population density.
5. Green Fluorescent Protein (GFP) is a common reporter to see whether the transformation and translation have been successful.
6. Protein E (lysis protein) is an operon from lambda bacteriophage dedicated to killing bacteria. It is expressed under the viral promoter and needs an additional ori site. [6]
7. There will be kanamycin resistance genes. Since any synthetic plasmid poses a metabolic burden on cells, the cell needs some "useful" gene to be interested in keeping the plasmid. [7]

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/plasmid-1-final.png)

**Figure 7.** The very first design of the plasmid

**Improve:**
Construct a plasmid that achieves our objectives and could be synthesized in situ with two commercially available plasmids.

**Build and Test:**
Feedback from our PIs and mentors (Prof. Ivan Vorobyev and Dr Aleena Saidova, PhD). Also, Logistic issues were a big factor due to impossibility of constructing and ordering plasmid and getting it on time. We decided on in situ synthesis via restriction cloning from two commercially plasmids.

**Learning and Optimization**
1. We need to simplify the construction of the plasmid, since it is very heavy. In situ synthesis is proposed within two commercially available addgene plasmids: L-Lactate inducible promoter, ColE1 part
2. GFP has to be present in very high concentrations, which is impossible when many genes are of interest. [8]
3. No AHL, No kill Switch. The restriction cloning, Golden Gate assembly, and PCR cloning are inefficient with such a large plasmid.

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/plasmid-2.png)

**Figure 8.** The in situ construction of plasmid of interest

## 6th Cycle Cohort

---

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/447.png)

**Figure 9.** Raw plasmid design

**Improve:**
Order a construct from a GenScript similar to an initial design.

**Build and Test:**
Logistic issues, lack of restriction enzymes, suggestion from our sponsor Dr Bolat Sultankulov. iGEM competition allows sponsorships from iDT, Twist, GenScript and allows to order ready constructs from biobricks.

**Learning and Optimization:**
1. ALPaGA operon instead of wild type lldRPD. Wild-type does not work in the tumor microenvironment, which is anoxic and has a high amount of lactate and glucose present. It only works in the presence of oxygen and the absence of glucose. It is not applicable for our project since we need anti-tumour drugs to be synthesized in the tumor conditions. [9]
2. Backbone changed to pET 9a, containing "rop" gene and no T7 promoter, which makes the plasmid a low copy. These changes stabilize the structure genetically by removing metabolic burden.
3. Addition of regulatory gene protein. Promoters can not be regulated without lldR, which blocks it in the absence of L-lactate. It is not present in the natural environment or bacterial media, so bacteria itself should synthesize it [4]
![](https://static.igem.wiki/teams/4782/wiki/design/project-design-1-1.png)

**Figure 10.** Initiation of transcription in presence of L-lactate.

4. Remove kill switch, and remove LuxI. There are too many genes for a bacteria to carry. It poses a high metabolic burden and will not be carried by bacteria.

![](https://static.igem.wiki/teams/4782/wiki/engineering-success/446.png)

**Figure 11.** Final Construct

References

1. Qin, H., Wang, J., Wang, T., Gao, X., Wan, Q., & Pei, X. (2018). Preparation and Characterization of Chitosan/β-Glycerophosphate Thermal-Sensitive Hydrogel Reinforced by Graphene Oxide. Frontiers in chemistry, 6, 565. https://doi.org/10.3389/fchem.2018.00565

2. National Research Council (US) Steering Group for the Workshop on Size Limits of Very Small Microorganisms. Size Limits of Very Small Microorganisms: Proceedings of a Workshop. Washington (DC): National Academies Press (US); 1999. Correlates of Smallest Sizes for Microorganisms. Available from: https://www.ncbi.nlm.nih.gov/books/NBK224751/

3. S Jimmy Budiardjo, Jacqueline J Stevens, Anna L Calkins, Ayotunde P Ikujuni, Virangika K Wimalasena, Emre Firlar, David A Case, Julie S Biteen, Jason T Kaelber, Joanna SG Slusky (2022) Colicin E1 opens its hinge to plug TolC eLife 11:e73297

4. Aguilera L.; Campos E.; Giménez R.; Badía J.; Aguilar J.; Baldoma L. Dual Role of LldR in Regulation of th.e lldPRD Operon, Involved in l -Lactate Metabolism in Escherichia coli. J. Bacteriol. 2008, 190, 2997–3005. 10.1128/jb.02013-07

5. Churchill ME, Sibhatu HM, Uhlson CL. Defining the structure and function of acyl-homoserine lactone autoinducers. Methods Mol Biol. 2011;692:159-71. doi: 10.1007/978-1-60761-971-0\_12. PMID: 21031311; PMCID: PMC3425365.

6. W. Lubitz et al. "Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174." Journal of Bacteriology (1984): 385-387

7. Plasmids 101: Antibiotic Resistance Genes, Marcy Patrick, 204 https://blog.addgene.org/plasmids-101-everything-you-need-to-know-about-antibiotic-resistance-genes

8. Lorang, J. M., Tuori, R. P., Martinez, J. P., Sawyer, T. L., Redman, R. S., Rollins, J. A., Wolpert, T. J., Johnson, K. B., Rodriguez, R. J., Dickman, M. B., & Ciuffetti, L. M. (2001). Green fluorescent protein is lighting up fungal biology. Applied and environmental microbiology, 67(5), 1987–1994. https://doi.org/10.1128/AEM.67.5.1987-1994.2001

9. Zúñiga, A., Camacho, M. D., Chang, H., Fristot, E., Mayonove, P., Hani, E., & Bonnet, J. (2021). Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synthetic Biology, 10(12), 3527–3536. https://doi.org/10.1021/acssynbio.1c00456

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# Experiments

## Experiments

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During our wet lab part of CellCare, we have used, created, and optimized **multiple protocols**. As this part is over, we realize how accurate and organized protocols are essential for successful lab work. Therefore, we collected all our protocols in two sets as another form of **contribution to the iGEM community!**

Below are the two documents with protocols. The first one, ***Hydrogel***, is created specifically for work with hydrogels, including experiments for gel structure optimization and characteristics determination. The second one, ***Cells***, is a typical list of protocols to plan and prepare for experiments with bacterial and mammalian cells (as our proof of concept).

**We hope that these documents will be helpful for other upcoming iGEM teams, and we wish the best of luck for them!**

Hydrogel

Cells

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* Content
* [Summary](#Summary)
* [Hydrogel synthesis](#Hydrogel)
* [Rheometer test](#Rheometer)
* [SEM images](#sem)
* [Bacterial release test](#release)
* [MTT assay](#mtt)
* [In vitro erosion test](#erosion)
* [Plasmid simulations](#plasmid)
* [References](#References)

# Results

## Summary

---

- Throughout our project, we synthesized temperature-sensitive in situ forming Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel.
- Solidification of the hydrogel occurs at 36.62 °C.
- Pore size depends directly on the amount of hyaluronic acid used during the preparation. The pore size of Chitosan/β-glycerophosphate hydrogel is about 56.61 ± 18.82 μm. In 0.5% HA hydrogel, the average pore size equals 18.39 μm, while 11.54 μm pores were observed when 1% HA was added. Pores do not form when 3% or more HA is used. In the hydrogel chosen for our system, 2% HA is added, and the average pore size is about 3.94 μm.
- The hydrogel can efficiently retain bacterial cells after encapsulation without allowing them to migrate freely to the outside environments. It does not have a severe negative effect on bacterial growth and enables their proliferation.
- In Vitro Erosion Test results indicate that the hydrogel can be sustained in the injection site for over 21 days.
- Due to unexpected logistics problems, we could not conduct bacterial transformation and further analysis as the plasmid still needed to be delivered to our team. However, we conducted virtual simulations of agarose gel electrophoresis and SDS-PAGE gel electrophoresis to determine the expected results of these experiments.
- After the plasmid arrives, we will check its performance in the lab according to our experimental design.

## Hydrogel synthesis and basic sol-gel transition evaluation

---

One of the key desired properties of the hydrogel for our system is temperature sensitivity. After several trials with different compositions, we synthesized an in situ forming a hydrogel. As demonstrated in Figure 1, the initial formulation (chitosan/glycerol) did not exhibit temperature sensitivity as the gels remained liquid after prolonged exposure to body temperature. As a result, we had to continue synthesis with another composition. Figures 2-4 illustrate the physical state of Chitosan/β-glycerophosphate hydrogel. Initially, the gel appeared to be a viscous liquid (Figure 2). After putting the hydrogel into the water bath at 37°C for 8 min, the gel became more dense and less transparent. Holding the gel in a water bath for 10 min resulted in the complete solidification of the hydrogel (Figure 3). Afterward, the gel was placed on the bench (room temperature ~25°C). The gel slowly returned to the liquid form after 40 min in these conditions. Figure 4 demonstrates the physical state of the gel after 40 min on the bench. Therefore, the Chitosan/β-glycerophosphate mixture produces an in situ forming temperature-sensitive hydrogel.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-1-results-of-hydrogel.gif)

**Figure 1.** Chitosan/Glycerol hydrogels after 20 min in the water bath at 37°C

![](https://static.igem.wiki/teams/4782/wiki/results/figure-2-chitosan-glycerophosphate-hydrogel-right-after-the-synthesis.jpeg)

**Figure 2.** Chitosan/β-glycerophosphate hydrogel right after the synthesis

![](https://static.igem.wiki/teams/4782/wiki/results/figure-3-chitosan-glycerophosphate-hydrogel-after-10-min-in-the-water-bath-at-37-c-1.jpeg)

**Figure 3.** Chitosan/β-glycerophosphate hydrogel after 10 min in the water bath at 37°C

![](https://static.igem.wiki/teams/4782/wiki/results/figure-4-chitosan-glycerophosphate-hydrogel-after-40-min-on-the-bench-25-c.jpeg)

**Figure 4.** Chitosan/β-glycerophosphate hydrogel after 40 min on the bench (25°C)

For pore size adjustment, hyaluronic acid was added to the mixture. Figures 5-6 demonstrate the physical states of Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel. Right after the synthesis, the gel could freely flow upon tube inversion (Figure 5). Subsequently, the hydrogel was placed into the incubator set to 37°C. As can be observed in Figure 6, the sample became completely solid after 8 min. Rheometric analysis must be performed for a more precise determination of gelation time and temperature.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-5-chitosan-right-after-synthesis.gif)

**Figure 5.** Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel right after the synthesis

![](https://static.igem.wiki/teams/4782/wiki/results/figure-6-hydrogel-after-8-min-in-the-static-incubator-at-37-c.gif)

**Figure 6.** Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel
 after 8 min in the static incubator at 37°C

## Rheological measurements

---

Rheological measurements were conducted to make precise conclusions regarding the solidification temperature and to evaluate how hydrogel deforms in response to applied forces (flow behavior). The storage modulus describes the elastic properties of the hydrogel, while the viscosity properties are characterized by the mechanical loss modulus [3]. The temperature sweep test with Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel solution between 5 and 50 °C showed a sol–gel transition behavior at 36.62 °C (Figure 7). When the temperature increased to 50 °C, the elastic modulus increased to 43.913 Pa. Both the storage modulus (G′) and loss modulus (G″) decreased slightly from 8.7 °C to almost 30.2 °C. Then, the storage modulus began to increase gradually, confirming the hydrogel's thermosensitive behavior [4]. The intersection point of G' and G" (tan δ: G"/G' = 1) represents the hydrogel solution's incipient gel point or the sol-gel transition. At a moment of 36.62 °C, it gets solidified. For practical purposes, a liquid is typically considered viscous if tan > 100:1 = 100, whereas a solid is considered ideally elastic if tan 1:100 = 0.01.

Steady shear flow (flow curves) report the dependency of the viscosity of the hydrogel on the applied shear rate [5]. This characteristic of hydrogels is advantageous for therapeutics delivery, tissue engineering, and other biomedical applications. The viscosity of the hydrogel stood at 88895E+05 mPA\*s and then started to decline till 1076mPa\*6. This demonstrated that applying the shear rate up to 100 1/s decreases the viscosity of the gel (Figure 10).
![](https://static.igem.wiki/teams/4782/wiki/results/figure-7-rheological-behavior-of-hydrogel-injection-storage-modulus-g-and-loss-modulus-g-of-the-hydrogel-injection-as-a-function-of-temperature.png)

**Figure 7.** Rheological behavior of hydrogel injection storage modulus (G′) and loss modulus (G″) of the hydrogel injection as a function of temperature.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-8-incipient-gel-point-or-sol-gel-transition-of-hydrogel.png)

**Figure 8.** Incipient gel point or sol-gel transition of hydrogel

![](https://static.igem.wiki/teams/4782/wiki/results/figure-9-the-viscosity-curve-of-the-chitosan-glycerophosphate-hyaluronic-acid-hydrogel-within-increasing-shear-rate-from-0-1-to-100-1-s.png)

**Figure 9.** The viscosity curve of the Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel
 within increasing shear rate from 0.1 to 100 1/s.

## SEM Imaging of hydrogels

---

Hydrogel pores must be tiny enough to encapsulate bacterial cells efficiently and prevent their uncontrolled migration to the outer environments [6]. The pores must also be large enough to permit macromolecule exchange between the encapsulated bacteria and the surrounding tumor microenvironment. The average pore size of Chitosan/β-glycerophosphate hydrogel equals 56.61 ± 18.82 μm [7]. It is too large for efficient trapping of E. coli cells since such big pores will enable bacterial migration from the gel. Initially, we added 4% (w/w) SH to the CH/GP hydrogel, intending to decrease the pore size. SEM images of Chitosan/β-glycerophosphate/Sodium Hyaluronate (4%) can be found in Figure 10. The average pore size was equal to 27.822 µM. This reduction is notable yet insufficient. As a result, we proceeded with further pore size optimization by adding different percentages of hyaluronic acid.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-10-sem-imaging-of-chitosan-glycerophosphate-sodium-hyaluronate-4-hydrogel-a.jpg)
![](https://static.igem.wiki/teams/4782/wiki/results/figure-10-sem-imaging-of-chitosan-glycerophosphate-sodium-hyaluronate-4-hydrogel-b.jpg)
![](https://static.igem.wiki/teams/4782/wiki/results/figure-10-sem-imaging-of-chitosan-glycerophosphate-sodium-hyaluronate-4-hydrogel-c.jpg)

**Figure 10.**  Rheological behavior of hydrogel injection storage modulus (G′) and loss modulus (G″)
 of the hydrogel injection as a function of temperature.

As shown in Table 1, the same percentage of hyaluronic acid led to a drastic reduction of the pore size, so we did not see any of them on SEM Images of the gel (Figure 15). Therefore, cross-linking between sodium hyaluronate and β-glycerophosphate is lower than between hyaluronic acid and β-glycerophosphate. As demonstrated in Figure 15, Chitosan/β-glycerophosphate/Hyaluronic acid (4% w/w) hydrogel has a film-like appearance. We did not observe any pores, even at 5.00 K X magnification. It is possible that the addition of hyaluronic acid (4% w/w) reduced the pore size to a nanometer scale. However, it was impossible to test this hypothesis because when we aimed for greater magnification, the gel began to change its morphology and form bubbles due to electron bombardment of a small area. We can conclude that hyaluronic acid, even in relatively small quantities, can significantly reduce the pore size of the Chitosan/Glycerophosphate hydrogel. Lack of pores would substantially reduce (and possibly eliminate) bacterial proliferation since macromolecule exchange would be inhibited in such gels. No pores were observed on Chitosan/β-glycerophosphate/Hyaluronic acid (3% w/w) hydrogel (Figure 14).

**Table 1.** Correlation of the pore sizes of CH/GP/HA hydrogels to the proportion of hyaluronic acid.

| CH proportion | GP proportion | HA proportion | Total DI Water | Average pore size |
| --- | --- | --- | --- | --- |
| 60 mg | 300 mg | 0 mg | 2.5 mL | 56.61 ± 18.82 μm [7] |
| 60 mg | 300 mg | 1.875 mg (0.5% w/w) | 2.5 mL | 18.39 μm |
| 60 mg | 300 mg | 3.75 mg (1% w/w) | 2.5 mL | 11.54 μm |
| 60 mg | 300 mg | 7.5 mg (2% w/w) | 2.5 mL | 3.94 μm |
| 60 mg | 300 mg | 11.25 mg (3% w/w) | 2.5 mL | Not Observed |
| 60 mg | 300 mg | 15 mg (4% w/w) | 2.5 mL | Not Observed |

The results of the SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (0.5% w/w) hydrogel are illustrated in Figure 11. The average pore size of this hydrogel is equal to 18.39 μm. Figure 12 contains the SEM image of Chitosan/β-glycerophosphate/Hyaluronic acid (1% w/w) hydrogel, which has an average pore size of 11.54 μm. Hence, these two formulations were eliminated from further experiments as the pores could allow high rates of bacterial migration.
Figure 13 demonstrates SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (2% w/w) hydrogel. The average pore size of this hydrogel is equal to 3.94 μm. So far, this data was the most fitting for the sustainment of the objectives of our system. Because of this, this formulation was chosen for further studies and bacterial encapsulation. To evaluate whether this hydrogel would minimize bacterial leakage, the Bacterial Release Test was performed.
Other hydrogels containing different percentages of hyaluronic acid between 2% and 3% could be synthesized in future experiments to select the most optimal formulation.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-11-sem-imaging-of-chitosan-glycerophosphate-hyaluronic-acid-0-5-w-w-hydrogel-jpg-page-0001.jpg)

**Figure 11.** SEM Imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (0.5% w/w) hydrogel.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-12-sem-imaging-of-chitosan-glycerophosphate-hyaluronic-acid-1-w-w-hydrogel.jpg)

**Figure 12.** SEM Imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (1% w/w) hydrogel.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-13-sem-imaging-of-chitosan-glycerophosphate-hyaluronic-acid-2-w-w-hydrogel.jpg)

**Figure 13.** SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (2% w/w) hydrogel.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-14-sem-imaging-of-chitosan-glycerophosphate-hyaluronic-acid-3-w-w-hydrogel.jpg)

**Figure 14.** SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (3% w/w) hydrogel.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-15-sem-imaging-of-chitosan-glycerophosphate-hyaluronic-acid-4-w-w-hydrogel-a.jpg)
![](https://static.igem.wiki/teams/4782/wiki/results/figure-15-sem-imaging-of-chitosan-glycerophosphate-hyaluronic-acid-4-w-w-hydrogel-b.jpg)

**Figure 15.** SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (4% w/w) hydrogel.

## Bacterial release test

---

One of the key reasons the system includes hydrogel is that the gel’s matrix can trap bacteria inside and prevent the uncontrolled spreading of the cells in the organism. To evaluate whether Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel can efficiently encapsulate bacteria and prevent their release into the surroundings, the Bacterial Leakage Test was conducted.
The primary (raw) data of the Bacterial Release Test can be found on the Lab Notebook page, while the calibrated results are summarized in Table 2. The first measurement (1 minute after sample loading) was taken as the baseline for the hydrogel-encapsulated bacteria sample, and the rest of the data from this group was normalized accordingly. The graph demonstrating the change in the OD600 nm of free E. coli cells and Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel encapsulated E. coli cells can be found in Figure 16. As illustrated, E. coli cells could rapidly grow and increase in the media as free bacteria. However, encapsulation of the E.coli cells into Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel efficiently prevented the bacteria migration out of the hydrogel. It is essential to note that bacterial release was not observed even at longer elapsed time points (24 hours, 48 hours, and 72 hours).

**Table 2.** Bacterial release test calibrated data.

|  | Sample |
| --- | --- |
| Elapsed Time (hh:mm:ss) | Encapsulated E.coli | Free E.coli |
| 00:01:00 | 0 | 0.0169 |
| 00:05:00 | 0.0222 | 0.0184 |
| 00:10:00 | 0.0645 | 0.0211 |
| 00:30:00 | 0.0092 | 0.0423 |
| 01:00:00 | 0.0362 | 0.0897 |
| 03:00:00 | 0.0646 | 0.3948 |
| 04:00:00 | 0.0997 | 0.4883 |
| 06:30:00 | 0.1364 | 0.8539 |
| 14:00:00 | 0.1735 | 1.3961 |
| 24:00:00 | 0.1646 | 1.633 |
| 48:00:00 | 0.1655 | 1.8415 |
| 72:00:00 | 0.1124 | 1.8831 |

![](https://static.igem.wiki/teams/4782/wiki/results/figure-16-graph-demonstrating-calibrated-results-of-the-bacterial-release-test.jpg)

**Figure 16.** Graph demonstrating calibrated results of the bacterial release test.

Overall, the results of this experiment demonstrate that Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel can efficiently encapsulate bacteria and minimize their escape to nearby environments. Therefore, integrating such systems could reduce the side effects of conventional bacterial therapies by preventing the unwanted spread of the cells across the organism.

## MTT Assay

---

Literature sources indicate that chitosan-based hydrogels exhibit biocompatibility and biodegradability, which allows us to view their injection into the human body as safe [1, 2]. However, the data regarding the cytotoxic effects of Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel on bacterial cells was not found. Hence, an MTT Assay was conducted to evaluate whether this formulation is suitable for bacterial encapsulation. This test is necessary since, in the case of high cytotoxicity of the gel to bacteria, it cannot be used in combination with bacterial therapeutics as most of the cells will not survive, and, as a result, the payload for treatment will be minimal.
Table 3 represents calibrated MTT Assay results. Raw data can be found on the Lab Notebook page. Figure 17 illustrates calibrated results in the form of box plots. The main principle of MTT Assay is based on the reaction in which nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductases reduce yellow 3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent) to a formazan, which forms insoluble purple granules. Hence, this method is applied to evaluate cell viability under specific conditions (such as the presence of certain chemicals). The first column (Bacteria/LB sample) was used as a positive control in this experiment. The second column contained Bacteria/Chloramphenicol (bacteriostatic antibiotic) and served as a negative control. The last two columns were filled with LB broth and empty hydrogel. 3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide was also added to these columns. The results of the controls satisfied our expectations since the positive control demonstrated a proper reduction of the MTT reagent by living cells. In contrast, the negative controls showed that in the absence of cells, the MTT reagent remains yellow, and the degree of color change (and, consequently, OD570 nm value) is proportional to the number of viable cells in the sample.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-17-box-plot-representation-of-calibrated-mtt-assay-data.jpg)

**Figure 17.** Box plot representation of calibrated MTT Assay data.

**Table 3.** MTT Assay calibrated data.

| Sample | Bac/LB | Bac/Chl | Bac/Gel |
| --- | --- | --- | --- |
| A | 1.680740875 | 0.765178875 | 0.56598775 |
| B | 1.362780875 | 0.973510875 | 1.12236775 |
| C | 1.440980875 | 0.726283875 | 1.07879775 |
| D | 1.414380875 | 0.790633875 | 0.86929775 |
| E | 1.401720875 | 0.672601875 | 1.05484775 |
| F | 1.247490875 | 0.895230875 | 0.98855775 |
| G | 1.069670875 | 0.973860875 | 0.99048775 |
| H | 1.2202709 | 0.826714875 | 2.02034775 |
| Mean | 1.373449 | 0.828002 | 1.0863365 |

As can be observed in Figure 17, the box plot of Bac/Gel samples is stretched. This is because the hydrogel consistency is not homogeneous, with some small clumps that form upon solidification. Photometric measurements were set to 570 nm. The calibrated mean optical density of the positive control (mean OD of LB was used as a blank) is equal to 1.373449, while the value for E.coli/Hydrogel samples (mean OD of hydrogel was used as a blank) is 1.0863365. Therefore, Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel affects bacterial viability by reducing it to 20.9045%. Hence, we can conclude that Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel is suitable for bacterial encapsulation as it does not demonstrate a significant adverse effect on bacterial proliferation, and its cytotoxicity is within acceptable range.

## In vitro erosion test

---

All reagents used to prepare Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel are biodegradable and biocompatible. For instance, chitosan degradation by nonspecific enzymes (lysozyme mainly) leads to the formation of simple polysaccharides, which can be excreted naturally or incorporated into the organism's metabolic pathways. The In Vitro Erosion Test was conducted to evaluate the rate of in vitro degradation of Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel. The results are summarized in Table 4 and Figure 18. As indicated in the graph, after 21 days, hydrogel mass decreased by 19.73 %. As the gel incubation time in PBS increases, the mass reduction rate increases. The experiment must be conducted for a prolonged period to evaluate the time required for a complete erosion.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-18-in-vitro-erosion-rate-of-chitosan-glycerophosphate-hyaluronic-acid-hydrogel.jpg)

**Figure 18.** In Vitro Erosion rate of Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel

**Table 4.** In Vitro Erosion Test results for Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel.

| Days in PBS | Initial Weight (g) | Final Weight (g) | Remaining % weight |
| --- | --- | --- | --- |
| 3 | 2.15 | 2.10 | 97.67 |
| 7 | 2.13 | 2.04 | 95.77 |
| 14 | 2.26 | 1.96 | 86.73 |
| 21 | 2.18 | 1.75 | 80.27 |

## Plasmid simulations

---

Our team encountered difficulties in plasmid import due to external factors, so we could not perform plasmid purification, bacterial transformation, protein synthesis and purification in practice. Thus, we decided to simulate plasmid agarose electrophoresis and Colicin E1 SDS-PAGE gel electrophoresis using specialized software.

Agarose gel electrophoresis results shown in Figure 19 were obtained through SnapGene software [8], dedicated to the design of plasmid and virtual simulations of it. The plasmid was preliminary run at 1.0% agarose gel electrophoresis, with 1st good showing linear plasmid and 2nd demonstrating its supercoil formation. The expected results should show both DNA bands in one well. However, the limitations of SnapGene did not allow us to perform such a simulation.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-19-agarose-gel-electrophoresis-of-plasmid.png)

**Figure 19.** Agarose gel electrophoresis of plasmid

The virtual simulation of SDS-PAGE gel electrophoresis was performed using ECEP2D learning software [9], which produces gel electrophoresis images using a preliminarily inserted protein amino acid sequence. The amino acid sequence was taken from the plasmid designed by our team. As can be observed from Figure 20, the expected band at 57 kDa is seen, which stands for colicin E1.
The mandatory requirement of ECEP2D software is to run an SDS-PAGE gel electrophoresis simulation with protein being digested by enzymes present in the list of the software. This obstacle was bypassed by choosing an enzyme that does not cleave the protein of interest. In our case, we used Trypsin M, which did not cut the sequence of colicin E1 we entered.

![](https://static.igem.wiki/teams/4782/wiki/results/figure-20-sds-page-of-colicin-e1.jpeg)

**Figure 20.** SDS-PAGE of Colicin E1.

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7. Qin H, Wang J, Wang T, Gao X, Wan Q, Pei X. Preparation and Characterization of Chitosan/β-Glycerophosphate Thermal-Sensitive Hydrogel Reinforced by Graphene Oxide. Front Chem. 2018 Nov 22;6:565. doi: 10.3389/fchem.2018.00565. PMID: 30555817; PMCID: PMC6282227.

8. Llc, G. B. (n.d.). SnapGene | Software for everyday molecular biology. SnapGene. https://www.snapgene.com/

9. Mayes, H., & Wong, C. F. (2018). Program for simulating gel electrophoresis of Enzyme-Digested proteins. Journal of Chemical Education. https://doi.org/10.1021/acs.jchemed.8b00225

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