Cycle 1 cohort: Adjustment of the general plan
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:
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:
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:
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.
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]
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.
6th Cycle Cohort
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]
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.
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
