Production of E. coli stock of the pUC57_FGF plasmid

(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.

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:

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

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.

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.

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.

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.

> 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.

> 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.

> 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

Martin Fisher, F., & Maratos Flier, E. (2016). Understanding the Physiology of FGF21. Annual Review of Physiology, 78, 223–241. https://doi.org/10.1146/annurev-physiol-021115-105339

Oost, L., Kustermann, M., Armani, A., Blaauw, B., & Romanello, V. (2019). Fibroblast growth factor 21 controls mitophagy and muscle mass. Journal of Cachexia, Sarcopenia and Muscle, 10(3), 630–642. https://doi.org/10.1002/jcsm.12409

Stanley, S., & Buettner, C. (2014). FGF19: How gut talks to brain to keep your sugar down. Molecular Metabolism, 3(1), 3–4. https://doi.org/10.1016/j.molmet.2013.10.008

Talukdar, S., & Kharitonenkov, A. (2021). FGF19 and FGF21: In NASH we trust. Molecular Metabolism, 46. https://doi.org/10.1016/j.molmet.2020.101152