The Engineering Cycle
The engineering cycle is a series of steps carried out in order to find a solution to a problem. It is important to define the problem and brainstorm ideas before creating a prototype test, that is then modified and improved until the solution meets the needs of the engineers project.
In the context of synthetic biology, which emphasizes the manipulation of DNA, genetic circuits, and biology systems, the engineering cycle highlights the importance of experimental design, computational modeling, and bioinformatics analysis. The 4 steps in the engineering cycle therefore provide a framework in design optimization in synthetic biology.
Design: identify the desired genetic circuit or biological system, design and assemble the necessary DNA components.
Build: synthesize the required DNA components, assemble the genetic circuits, and insert them into the appropriate host organism or chassis.
Test: characterize the behavior and performance, measuring outputs and evaluating against design specifications.
Learn: validate the result and gain insights from the analysis to make improvement.
What have we done?
1. FGF in Lactobacillus casei (BBa_K5402016 to BBa_K5402019)
L. casei is a gram-positive, non-spore-forming, rod-shaped bacterium, commonly found in the gastrointestinal tract of humans, as well as in fermented dairy products and plant-based foods, such as yogurt, cheese, and kimchi.
L. casei is also widely used as a probiotic, as it has been shown to provide various health benefits, such as modulation of the immune system, reduction of inflammation, improvement of gut health and digestion and potential reduction of the risk of certain diseases, such as diarrhea, cancer, and allergy.
One of the very commonly known fermented dairy drinks that contains the probiotic bacterium, Yakult, contains Lactobacillus casei Shirota. This stain is specifically selected and cultivated for its ability to survive the acidic environment of the human gastrointestinal tract. Yakult is primarily marketed for its potential health benefits, which are attributed to the presence of the L. casei Shirota strain. Studies suggest that this probiotic strain helps improve gut health and digestion, enhance immune system function, and reduce the risk of certain gastrointestinal disorders.
Moreover, L. casei has a relatively versatile metabolism and can utilize a wide range of primary bile acid to produce secondary bile acids, the primary substrate to the FXR receptor. In which, FXR receptors are activated upon substrate binding to upregulate genes including FGF19 and FGF21, which plays an important role in the context of obesity and metabolic disorders. (Detailed explanation can be found in “Project Description” and “Results”)
Integrating all the ideas, we were sparked to make use of L. casei as a vector with pTRKH3 plasmid used to carry our target genes (FGF19 and FGF21). We believe it would be a perfect candidate for “oral drug”.
After constructing the plasmid carrying FGF genes and transforming it into L. casei, we test the result with gel electrophoresis, Sanger sequencing, and Western Blot. With positive results obtained, we confirm that L.casei is said to be a suitable FGF carrying vector. (results can be seen in the “Results” page)
2. Modifications on FGF genes (BBa_K5402000 to BBa_K5402003)
There are known to be certain limitations in the natural FGF proteins products, which include poor pharmacokinetic and biophysical properties. Individuals with obesity might even develop systemic and/or adipose depot-selective FGF21 resistance and underpin insulin resistance (Geng et al., 2020). As a result, modifications were applied to improve their stability, half-life, membrane solubility or resistance against degradation.
First of all, methionine and cysteine are extremely sensitive to almost all forms of reactive oxygen species, which makes them antioxidative (Bin et al., 2017). Methionine and Cysteine residues can therefore be replaced with non-oxidizable amino acids like Alanine or Serine to prevent oxidative damage.
Besides, we took reference from a journal article to perform modifications on FGF19. It is proved that the modified FGF19 has the ability to regulate bile acid metabolism by inhibiting key bile acid synthesis enzymes; to attenuate liver inflammation, fibrosis, and restored bile acid homeostasis, comparable to the effects of FGF19WT; is protected against cholestatic liver injury by reducing bile acid levels, inflammation, and liver damage; does not induce proliferation or activate oncogenic signalling pathways (Shi et al., 2023).
Lastly, N-glycosylation engineering is also proven to enhance protease resistance and solubility (Weng et al., 2018).
In order to ensure the modifications would not be damaging the protein product formed, their functional effects on the protein were predicted using SIFT and PolyPhen-2. If the score > 0.05, the modification can be tolerated.
From “Results”, similar expressions between the natural and modified FGF genes can be observed. Thus, the modifications are said to be successful, in which the production of the FGF proteins is not adversely affected.
3. Insertion of miRNAs into plasmid backbone (BBa_K5402020)
RNA interference (RNAi) was coined to describe a cellular mechanism that uses the gene's own DNA sequence to turn it off, a process that researchers call silencing. It is a natural biological process that regulates gene expression by “interfering” with messenger RNA (mRNA), which is responsible for translation and thus protein production.
Making use of this knowledge, scientists have been investigating the possibility of artificially induce RNAi to regulate other genes. One of the models makes use of a specific ACGT sequence. By cloning it into a plasmid, microRNA (miRNA) precursors can be delivered into a cell. After some processing, the miRNA is loaded into a protein complex called the RISC complex, which guides miRNA to mRNA. The miRNA-RISC complex then inhibits translation, which in turn inhibits gene expression.
Being inspired by the model, the genetic circuit of our plasmid was created by inserting 3 siRNA sequences into respective 166-bp pre-miR-155 backbone, consisting of a CMV promoter and pre-miRNA parts. The CMV promoter was used to express a miRNA precursor, and the miRNA sequence was substituted with siRNA. Exosomes were chosen due to their relative lack of side effects compared to naked miRNAs/siRNA or other delivery vehicles. For the selection of siRNAs, we identified certain genes and knockdown targets. To start off, we will target the MKK6 gene which is elevated in the white adipose tissue of obese individuals. Literature has shown that the deletion of MKK6 can increase energy usage and thermogenic capacity of one’s white adipose tissue, helping to protect against further development of obesity. In addition, MKK6 deletion also stimulates T3-stimulated UCP1 expression in adipocytes. This also increases thermogenic capacity. Hence, the MKK6 gene holds potential to alleviate obesity. We will also target the p53 gene. The p53 gene can modulate lipid metabolism and studies have found that p53 activation in obese mice and humans promotes the senescence of adipocytes and the recruitment of macrophages. Finally, the p65 gene was chosen as due to its role in the NF-kappaB pathway, which could possibly induce inflammation significantly.
4. Stable cell line producing adipose-targeted exosome
The HEK293 cells are immortalized human embryonic kidney cells, commonly used in biomedical research and were chosen due to their high efficiency and stability for transfection. This plasmid will be co- transfected to the HEK293 cells. This process of transfection will go on for 48 hours. After this, the exosome will be produced and secreted into the cell supernatant. Once again, we will wait for 48-72 hours, after which, puromycin will be added. Puromycin is a naturally derived antibiotic that hinders the production of proteins by inserting itself into the end of growing protein chains, preventing their further elongation and causing the translation process to end prematurely. Puromycin is added to our cells mainly to screen for stable infections as puromycin resistant HEK293 cells contain the DNA construct permanently incorporated into the cell genome. Following this, the cells can now stably produce the adipose-targeted exosome with miRNAs of interest.
Exosome characterization and functional analysis on adipocytes can be found on “Results”.
References
Bin, P., Huang, R., & Zhou, X. (2017). Oxidation Resistance of the Sulfur Amino Acids: Methionine and Cysteine. Biomed Res Int. https://doi.org/10.1155/2017/9584932
Geng, L., Lam, K., & Xu, A. (2020). The therapeutic potential of FGF21 in metabolic diseases: from bench to clinic. Nature Reviews Endocrinology, 16, 654-667. https://doi.org/10.1038/s41574-020-0386-0
Shi, L., Zhao, T., Huang, L., Pan, X., Wu, T., Feng, X., Chen, T., Wu, J., & Niu, J. (2023). Engineered FGF19ΔKLB protects against intrahepatic cholestatic liver injury in ANIT-induced and MDR2-/- miCe model. BMC Biotechnology, 23, 43. https://doi.org/10.1186/s12896-023-00810-9
Weng, Y., Ishino, T., & Sievers, A. et al. (2018). Glyco-engineered Long Acting FGF21 Variant with Optimal Pharmaceutical and Pharmacokinetic Properties to Enable Weekly to Twice Monthly Subcutaneous Dosing. Scientific Reports, 8, 4241. https://doi.org/10.1038/s41598-018-22456-w