iGEM 2024 — Team cuhk-hongkong-sbs

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# 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.

![](https://static.igem.wiki/teams/5402/engineering/the-engineering-cycle.png)

# What have we done?

![](https://static.igem.wiki/teams/5402/engineering/registry-5.png)

## 1. FGF in Lactobacillus casei (BBa\_K5402016 to BBa\_K5402019)

![](https://static.igem.wiki/teams/5402/engineering/lacto.png)

##### 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.

![](https://static.igem.wiki/teams/5402/engineering/yakult.png)

![](https://static.igem.wiki/teams/5402/engineering/fxr-receptor.png)
![](https://static.igem.wiki/teams/5402/engineering/secondary-bile-acids.png)

##### 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”](https://2024.igem.wiki/cuhk-hongkong-sbs/description) and [“Results”](https://2024.igem.wiki/cuhk-hongkong-sbs/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”.

![](https://static.igem.wiki/teams/5402/engineering/action-of-fxr.png)
![](https://static.igem.wiki/teams/5402/engineering/19.png)
![](https://static.igem.wiki/teams/5402/engineering/21.png)

##### 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”](https://2024.igem.wiki/cuhk-hongkong-sbs/results) page)

## 2. Modifications on FGF genes (BBa\_K5402000 to BBa\_K5402003)

![](https://static.igem.wiki/teams/5402/engineering/43.png)

##### 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).

![](https://static.igem.wiki/teams/5402/engineering/44.png)

##### 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”](https://2024.igem.wiki/cuhk-hongkong-sbs/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)

![](https://static.igem.wiki/teams/5402/engineering/model.png)

##### 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

![](https://static.igem.wiki/teams/5402/engineering/our-plan.png)

##### 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”](https://2024.igem.wiki/cuhk-hongkong-sbs/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>

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# Production of E. coli stock of the pUC57\_FGF plasmid

![](https://static.igem.wiki/teams/5402/results/puc57.png)

(from Genescript)

##### pUC57 is a commonly used plasmid cloning vector in E. coli. The vector length is 2,710 bp and is isolated from E. coli strain DH5-alpha by standard procedures. Therefore, the FGF genes were first inserted into this vector for quantitative production of the plasmid. Making use of the E. coli competent cells DH10B, transformation is then performed to generate Top10\_pUC57\_FGF.

![](https://static.igem.wiki/teams/5402/results/gel-1.png)

##### Figure 1. 1% agarose gel electrophoresis after colony PCR using SP common forward primer and FGF19/FGF21 reverse primer.

##### > FGF19 (642 bp), mFGF19 (642 bp), FGF21 (609 bp), and mFGF21 (633 bp) all gave a band near 650 bp. This indicates that transformation was successful.

# Transferring the desired genes into a designated expression vector pTRKH3

##### The plasmid pTRKH3, with a backbone length of around 7500 base pairs, is an expression plasmid compatible with Lactobacillus. To introduce the FGF genes into the pTRKH3, miniprep was first performed to obtain pUC57\_FGF from E. coli, followed by PCR to amplify the insert region (primer used: Universal MluI forward & iGEM reverse). By restriction digestion (restriction enzymes: MluI & BamHI), the FGF genes were isolated and purified using gel extraction techniques. After that, ligation was performed using T4 DNA ligase to generate pTRKH3\_FGF, which was then transformed into E. coli for selection.

process of pTRKH3\_FGF construction:

![](https://static.igem.wiki/teams/5402/results/plasmid-diagram.png)

##### In order to verify the transformed clones, apart from colony PCR, Sanger sequencing was also performed.

![](https://static.igem.wiki/teams/5402/results/gel-2.png)

##### Figure 2. 1% agarose gel electrophoresis after colony PCR using MluI forward primer and BamHI reverse primer.

##### > FGF19 (642 bp), mFGF19 (642 bp), FGF21 (609 bp), and mFGF21 (633 bp) all gave a band near 650 bp. This indicates that transformation was successful. (Mouse Fgf 15 and Fgf21 gene were also transformed. However, they failed in Sanger sequencing, we therefore excluded them for downstream experiments.)

# Transform L. casei with the genes

##### To transform L. casei with the genes, we first generate a L. casei shirota competent cell ATCC 393. Then, electroporation was applied for transformation to generate Shirota\_pTRKH3\_FGF.

##### In order to verify the transformed clones, apart from colony PCR, Sanger sequencing was also performed.

![](https://static.igem.wiki/teams/5402/results/gel-3.png)

##### Figure 3. 1% agarose gel electrophoresis after colony PCR using MluI forward primer and BamHI reverse primer.

##### > FGF19 (642 bp), mFGF19 (642 bp), FGF21 (609 bp), and mFGF21 (633 bp) all gave a band near 650 bp. This indicates that transformation was successful.

# Verification of the products

##### Western Blot is widely used for the detection and quantification of specific proteins. The use of specific antibodies allows for the detection of a particular target protein within a complex mixture, giving a high specificity. On the other hand, The signal intensity can be used to estimate the relative abundance of the target protein, while the separation of proteins by size also provides information about the molecular weight of the target protein. For our verification, proteins extracted from L. casei were used for Western blot analysis. His-tag primary antibody and anti-rabbit HRP-conjugated secondary antibody were used. The use of His-tag allows simple all-at-once detection of the target proteins. Here, we make use of Western Blot ONLY for protein detection, but not quantification.

![](https://static.igem.wiki/teams/5402/results/fgf-wb.png)

##### Figure 4. Western blot analysis showing the expression of FGF19, mFGF19, FGF21, and mFGF21 respectively, where two different clones of each gene were taken for analysis.

##### > Figure shows that all of the desired FGF proteins were successfully produced. On top of that, the modified FGF (mFGF) genes showed a similar expression pattern compared to the unmodified one. This reflects the modifications were successful and feasible.

##### > However, for all four FGF products, the bands shwon are around 5kDa shorter than expected. This may due to intrinsic digestion activity of the bacteria or accidental protein denaturation during extraction process. This situation could be improved by purifying the proteins before Western Blot analysis.

# Maintenance of HEK293 cell line

##### Human Embryonic Kidney (HEK) 293 cells cultured in DMEM supplemented with 10% Fetal Bovine Serum and incubated at 37°C with 5% CO2. Penicillin and Streptomycin at 1 μl/mL was added to culturing medium. Cell passage was conducted every 2-3 days at 70-80% confluency and passaged at a ratio of 1:8 to 1:10.

![](https://static.igem.wiki/teams/5402/results/cell-line.png)

# Differentiation and culture of white adipocytes

##### White adipocyte precursors were cultured on 12-well plates at 35-50K/well and maintained in 37°C incubators with 5% CO2. Cells were cultured with DMEM medium (company) supplemented with 10% Fetal Bovine Serum (Gibco). The cells were screened for fluroresence from the mCherry tag.

![](https://static.igem.wiki/teams/5402/results/mirna-1.png)

![](https://static.igem.wiki/teams/5402/results/mirna-2.png)

##### > Significant fluorescence was seen in the experimental group.

# Western Blot for exosome characterization

##### Next, we attempted to characterize the exosomes in both the cell lysate and cell culture medium. Western Blot was conducted on the cell lysates and supernatant to characterize the exosome.

![](https://static.igem.wiki/teams/5402/results/mirna-western-1.png)
![](https://static.igem.wiki/teams/5402/results/mirna-western-2.png)

![](https://static.igem.wiki/teams/5402/results/mirna-w3.png)

##### > Results were unfortunately inconclusive due to inconsistencies with protein size.

# Functional analysis on adipocytes: Quantitative Real-Time PCR

##### RNAiso Reagent (Takara) was used to extract total RNA according to the manufacturer’s protocol. cDNA was synthesized with 5X PrimeScript RT Master Mix kit (TaKaRa), and quantitative SYBR Green RT-PCR was then performed. Each sample was run with three technical replicates. RNA levels were normalized to that of FABP4.

![](https://static.igem.wiki/teams/5402/results/mirna-3.png)

![](https://static.igem.wiki/teams/5402/screenshot-2024-10-02-at-9-58-52-pm.png)

##### > From the qPCR results, we can see that there is a promising effect on our experimental group on inflammation markers. More repetitions will be needed to confirm the effects.

# Overall conclusion

##### From the PCR and Sanger sequencing results, L. casei is shown to be able to carry FGF genes. The modified FGF genes were then successfully translated, giving desired protein products, which can be concluded from the Western Blot result. FGF19, is a growth factor proved to help with obesity by acting on the liver to inhibit bile acid synthesis, promote glycogen synthesis, increase insulin sensitivity in peripheral tissues (Talukdar & Kharitonenkov, 2021), and also has effect on the brain to modulate appetite and energy expenditure so as to reduce food intake and increase energy expenditure (Stanley & Buettner, 2014). As for FGF21, it induces weight loss by regulating fatty acid oxidation and improving glycemia (Fisher & Flier, 2016). Besides, it is proved to cause browning and proceed with an anti-inflammatory effect (Geng et al., 2020). In addition, FGF21 is also highly associated with obesity in elderly. It is suggested that muscle and bone mass decrease with aging, while fat mass increases (Colleluori & Villareal, 2021). With dropping muscle mass, there is reduced place for glycogen storage and thus causes increase in blood glucose level. As a result, elderly generally face the problem of increased risk of obesity together with diabetes. As the desired FGF products are generated from L.casei, we therefore can conclude that L.casei possesses the ability to acquire anti-obesity properties. As shown in the results above, while human FGF19 and FGF21 succeeded, mouse Fgf15 and Fgf21 failed in Sanger sequencing after several rounds of attempts. Noted that FGF gene sequences are quite different in human and mouse, failure may due to the following reasons: 1. Codon Usage Bias: the codon usage pattern in the mouse Fgf genes may not be optimized for efficient expression in L. casei, which has a different codon bias compared to the mouse.This mismatch in codon usage can lead to reduced translation efficiency or even improper folding and instability of the mouse Fgf proteins in L. casei, resulting in the failure to detect the correct sequence. 2. Regulatory Sequence Differences: the promoter, ribosome-binding site, or other regulatory sequences used to drive the expression of the mouse Fgf genes may not be recognized or function properly in the L. casei host.This could result in poor or no expression of the mouse Fgf proteins, even if the gene is successfully transformed, making it difficult to detect the correct sequence. 3. Plasmid Stability and Replication Issues: the mouse Fgf plasmid may not replicate or be maintained as efficiently in L. casei compared to the human FGF plasmid.This could lead to the loss or instability of the plasmid, making it challenging to obtain the correct sequence information. 4. Transformation Efficiency Differences: the transformation efficiency for the mouse Fgf plasmid may be inherently lower compared to the human FGF plasmid, due to differences in plasmid size, DNA sequence, or other factors.This could result in fewer successful transformation events for the mouse Fgf plasmid, leading to the observed failure in Sanger sequencing. On the other hand, exosome packaged in HEK293T is hoped to deliver the three miRNAs to adipocytes, with the functions of controls T3-mediation of white adipose tissue, suppresses ESC proliferation, and inhibits induction of inflammatory cytokines respectively. The plasmid was successful transfected into cells as seen from the mCherry fluorescence results. Although there were inconclusive results obtained from Western due to inconsistencies with protein size, functional analysis on adipocytes via qPCR show promise in anti-inflammation and anti-senescence effects on white adipocytes. Our literature review found that Lactobacillus are able to secret exosomes (Liu, 2024). Therefore, at the end of our project, we aim at combining the two teams to synthesize anti-obesity exosomes and FGF carrying Lactobacillus.

# List of success

##### - Transformation of chemically-competent E. coli cells with plasmid DNA - Gel electrophoresis and gel clean-up of vector DNA and target gene fragments - Restriction digestion of vector and target genes with MluI and BamHI enzymes - Ligation of vector and target gene fragments using T4 DNA ligase - Colony PCR to screen for correct clones containing the target genes - Miniprep to isolate plasmid DNA from positive clones - Protein extraction from E. coli cells - Electroporation of competent L. casei cells with plasmids - Sonication to lyse E. coli cells for protein extraction - Western blot analysis to detect target protein expression - Maintenance of HEK293 cell culture - Differentiation and culture of white adipocytes

# List of failure

##### - Initial low DNA concentration after gel clean-up - Unsuccessful transformation with no colonies found on selection plates after multiple attempts - Inability to find the correct clone containing the FGF15 gene after repeated restriction digestion and transformation - Colony PCR showed no bands - Gel electrophoresis and gel clean-up steps had lower than expected DNA concentrations - Some transformation attempts did not yield any colonies on selection plates - Restriction digestion using wrong enzymes did not show the expected bands - Repeated failed colony PCR results for clones - Gel electrophoresis issues, with strange U-shaped bands and no clear bands - Miniprep using lysozyme did not improve colony PCR results - Western blot issues with the protein ladder not running properly - Protocol for adipocytes differentiation states to differentiate the cells until day 7 or 8. However, few lipid droplets were seen on day 7. - A change in cell morphology was seen in around 10 passages, which may affect experimental results. - Results of Western Blot for exosome characterization were unfortunately inconclusive due to inconsistencies with protein size. Solutions: 1. We repeated the experiments. 2. Since the electrophoresis keeps showing no bands, we first perform troubleshooting to identify and resolve any issues that may be preventing the successful amplification of the target DNA sequences. 3. However, the result is still not ideal. After several trials, we suspected to be problem with undesired plasmid quality and number of copy. Therefore, we repeated the experiment from bacteria culture using the glycerol stock.

# Future plan

##### 1. Detail functional analysis on the modified FGF genes. 2. Perform codon optimization for mouse Fgf genes. 3. Perform purification for the FGF protein products and repeat Western Blot. 4. The his-tag antibody used for Western Blot may associate with non-specific binding, we are suggusted to repeat Western Blot with specific FGF antibodies. 5. Animal testing and cell assays to verify the actual effect of the products on fighting against obesity, and confirm there would not be harmful side effects. 6. Perform mass spectrometry to ensure no toxic metabolites are produced by L.casei upon engineering. 7. Repeat experiments for miRNA teams.

# References

Colleluori, G., & Villareal, D. (2021). Aging, obesity, sarcopenia and the effect of diet and exercise intervention. Experimental Gerontology, 155. <https://doi.org/10.1016/j.exger.2021.111561>

Geng, L., Lam, K., & Xu, A. (2020). The therapeutic potential of FGF21 in metabolic diseases: from bench to clinic. Nature Reviews Endocrinology, 16, pages654-667. <https://doi.org/10.1038/s41574-020-0386-0>

Liu, R. (2024). A promising area of research in medicine: recent advances in properties and applications of Lactobacillus-derived exosomes. Frontiers in Microbiology, 15. <https://doi.org/10.3389/fmicb.2024.1266510>

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