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Engineering

Design

Issue of Interest: Pioneering Substitute for FBS

This year, we aimed to develop conditioned media as a potential substitute for fetal bovine serum (FBS) in cultured meat production. FBS has been a standard practice; however, it involves drawbacks in terms of ethical concerns, batch-to-batch variability, and potential transmission of pathogens. Therefore, we will focus on developing a sustainable and FBS-free growth medium for cultivating bovine muscle cells that could potentially replace FBS.

Purpose Behind the Design

The design of our machine, in part, was fueled by two proteins of interest: Afamin and Wnt3a, two crucial components that could support cell growth and proliferation without the need for FBS. We sought to engineer human embryonic kidney (HEK293) cells that stably contain and express Afamin and Wnt3a genes. Afamin is a glycoprotein that plays a significant role in binding and stabilizing various growth factors, while Wnt3a is a critical signaling molecule involved in cell proliferation and differentiation. The co-expression of these two factors was expected to create an ideal microenvironment for the growth of bovine muscle cells. HEK293 cells are commonly used for genetic engineering experiments due to their high transfection efficiency and robust growth characteristics. The transgenesis requires the following:

  • Integration of Afamin and Wnt3a genes into the host genome
  • Stable expression of Afamin and Wnt3a in a polycistronic mRNA-like manner
  • Formation of a stable Afamin-Wnt3a protein complex without fusing two proteins

We identified the PiggyBac Vector System as a plausible medium for fulfilling these conditions. PiggyBac Vector System is a powerful implement for introducing and stably integrating genetic material into host cells, ensuring long-term and stable expression of the desired genes.

[Background: PiggyBac Vector System]

The PiggyBac Vector System is comprised of two elements: PiggyBac Vector and Super PiggyBac Transposase Expression Vector. PiggyBac Vector contains genes of interest and is intervened by terminal inverted terminal repeat (ITR) sequences at its 5’ and 3’ ends. Super PiggyBac Transposase Expression Vector, self-explanatorily, expresses Super PiggyBac Transposase; transposases are enzymes that identify the terminal repeat sequences in a gene and bind DNA transposon between each of the terminals. Upon co-transfection of two vectors into the host cells, Super PiggyBac Transposase recognizes ITR sequences and thereby integrates the intervening sequence (containing the genes of interest) into the host genome at TTAA–a process known as ‘PiggyBac-ing.’ Overall, this system integrates the desired set of genes into the host genome.

Our first element of our PiggyBac Vector System is the BII-CMV-AfmW3A Vector (Part: BBa K4869000). Primarily, it contains Afamin (Part: BBa K4869004) and Wnt3a (Part: BBa K4869005) flanked by ITR sequences (Part: BBa K4869006). Our full genetic construct is shown in Figure 1, and our key elements (and their rationale) are as follows:


Figure 1 (Inverted Terminal Repeat (ITR) sequences are positioned at the 5’ and 3’ ends. CMV promoter, afamin, 2a, wnt3a, IRIS, puromycin resistance gene (PuromycinR), WPRE, and SV polyA elements are designed to transfer into HEK293 genomic DNA by Supper Piggybac transposase)
Figure 2 (2A self-cleaving peptides sequence used in this vector)
  • ITR sequence: The Super piggyBac transposase is a powerful tool for inserting genes into a host genome using the Inverted Terminal Repeat (ITR) sequences. The piggyBac transposon system operates through a "cut-and-paste" mechanism to move genetic material from one location to another within a genome. Transposase recognizes the ITR sequence and cuts and pastes it into HEK293 genomic DNA.
  • CMV promoter: The BII-CMV-AfmW3A vector's inclusion of the CMV promoter is a critical element in the design of this genetic construct. The CMV promoter, which stands for Cytomegalovirus promoter, is potent and widely utilized in molecular biology and genetic engineering. The CMV promoter is favored for its strong and constitutive (constant) expression activity in a broad range of mammalian cell types, making it an excellent choice for driving the expression of genes of interest. This promoter is known for its robustness and ability to initiate transcription efficiently.
  • Afamin: The mouse afamin gene was used and positioned after the CMV promoter.
  • 2A: The 2A peptide cleavage system is a powerful tool in molecular biology and biotechnology because it allows the co-expression of multiple genes from a single mRNA transcript. The 2A peptide sequence is typically placed between two genes of interest on a single mRNA transcript to produce two proteins. Therefore 2A peptide sequence is placed between Afamin and Wnt3a. The insertion of an 18-amino acid 2A self-cleaving peptide between Afamin and Wnt3a proteins enables co-expression in a single mRNA, offering a powerful and precise method for achieving stable protein expression (Figure 2).
  • IRES: IRES sequence in a DNA vector is a valuable tool for enabling the expression of multiple proteins from a single mRNA transcript. It allows for cap-independent translation initiation and co-expresses genes of interest without fusing them, making it a versatile element in molecular biology and genetic engineering. The IRES sequence was positioned between our target gene and the puromycinR gene.
  • PuromycinR: The puromycin resistance gene is commonly used in molecular biology and cell biology research as a selectable marker. Its purpose in the selection process is to enable the isolation and maintenance of cells or organisms that have been successfully transfected or transformed with a specific DNA construct. In our experiment, puromycin was used to isolate the stable cells that contain the Afamin and Wnt3a sequence in HEK293.
  • WPRE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence in a DNA vector is a regulatory element that enhances gene expression by increasing mRNA stability and facilitating efficient cytoplasmic accumulation. Its inclusion in vectors is a valuable strategy to boost and stabilize the expression of genes.
  • SV polyA: The SV40 polyadenylation sequence in a DNA vector is crucial for proper termination of transcription, mRNA stability, and efficient translation of the gene of interest. It ensures that the mRNA is processed correctly and is equipped for stable and productive expression within the host cell.
Figure 3 (Super PiggyBac Transposase Expression Vector contains the Super PiggyBac Transposase gene)

The second element of our System is the Super PiggyBac Transposase Expression Vector (Part: BBa K4869001, Figure 3). Upon the expression of Super PiggyBac Transposase, it recognizes the ITR sequences and integrates the flanked genetic construct into the genome of HEK293 cells. Accordingly, HEK293 cells, genomically, contain both Afamin and Wnt3a genes. This is preceded by lipofectamine-mediated co-transfection of the BII-CMV-AfmW3A Vector and Super PiggyBac Expression Vector into HEK293 cells (Figure 4).

Figure 4 (Engineered HEK293 cells producing Afamin and Wnt3a proteins into conditioned media)
Upon genomic integration, engineered HEK293 cells simultaneously express Afamin and Wnt3a genes in a polycistronic mRNA-like manner, while the nature of the integrated genetic construct prevents the fusion of these proteins. The resulting proteins will be secreted into the surrounding conditioned cell culture media. This Afamin-Wnt3a enriched media can then be collected and utilized in experiments involving the growth of bovine cells.
Figure 5 (Predicted Afamin-Wnt3a complex structure. Afamin in gray and Wnt3a in green)

Overall, we expect a complex between Afamin and Wnt3a proteins (Figure 5). Afamin is a serum glycoprotein that can maintain the active and water-soluble form of lipidated Wnt protein in hydrophilic environments. The experiments conducted by researchers at Osaka University and Keio University School of Medicine showed that afamin binds tightly to Wnt proteins, forming a 1:1 complex that remains soluble in aqueous buffer after isolation. This complex can induce signaling in various cellular systems, including the intestinal stem cell growth assay.

Parts for Implementation

To implement the function of our system, we will use the Parts (which we newly added to the Registry of Standard Biological Parts this year) listed below. Detailed descriptions and validation of each part can be further viewed on both Parts and Parts Collection pages.

Build

Overview: Building Process

[Part 1: Cloning BII-CMV-AfmW3A Vector and Polymerase Chain Reaction (PCR)]

Since we did not have enough BII-CMV-AfmW3a Vector (purchased from AddGene) for our experiment, we had to transform this plasmid vector into E.coli DH5alpha for amplification. The amplified plasmid was purified via a MiniPrep Kit. We conducted PCR afterward to ensure the presence of Afamin and Wnt3a genes in the purified vector. The following primer sequences were used in this experiment:

  • Afamin (250 bp)
    • Forward primer: 5′ - ATGAGACACTTAAAACTTACAGG - 3′
    • Reverse primer: 5′ - GAAGTGTGTTGTCAGCCCAGCAC - 3′
  • Wnt3a (200 bp)
    • Forward primer: 5′ - ATGGCCCCACTCGGATACTTCTTAC - 3′
    • Reverse primer: 5′ - GCCACGCTGGGCATGATCTCCA - 3′
Figure 6 (Amplified Afamin and Wnt3a DNA products by PCR are visualized using the agarose gel. Lane 1: standard marker, Lanes 2 and 3: vector plasmid #1, Lanes 4 and 5: vector plasmid #2, Lanes 6, 7, and 8: vector plasmid #3. Afamin targeting primers were used in Lane 2, 4, and 6. Wnt3a targeting primers were used in Lane 3, 5, 7, and 8)

A total of 8 samples were loaded into the agarose gel, and it was run for 20 minutes at 100 V in the agarose electrophoresis tank. Each vector plasmid had both Afamin and Wnt3a gene indicators, and the bands represented the amplified products. According to the result, vector #2 and #3 bands showed multiple bands, which demonstrated the possibility of any mutations or altercations during the purification process. On the other hand, vector #1 plasmid represented clear single bands in both genes: Afamin at 250 bp and Wnt3a at 200 bp (Figure 1). Therefore, we concluded that vector #1 plasmid was the most suitable for further experiments.

[Part 2: Validation of Engineered HEK293 Cells through Cell Morphology Analysis]

Figure 7 (Cell morphology of HEK293 cells of negative control, 1:2.5 transfected sample, 1:5 transfected sample)

Subsequently, we co-transfected the BII-CMV-AfmW3A Vector and Super PiggyBac Transposase Expression Vector into HEK293 cells via lipofectamine. As previously demonstrated, the Super PiggyBac Transposase recognizes our genetic construct in the BII-CMV-AfmW3A (flanked with ITR sequences) Vector–and integrates it into the HEK293 genome at TTAA sites. Such transgenesis, indeed, creates engineered HEK293 cells containing Wnt3a and Afamin genes–primary supplements of our conditioned media.

Three experimental conditions–the negative control, 1:2.5 transfected sample, and 1:5 transfected sample–show negligible differences in their cell morphology and structure (Figure 7). This, in fact, is a primary checkpoint in our research, for our procedures (e.g., puromycin treatment and co-transfection of vectors) may potentially impair the structural and cellular integrity of HEK293 cells. Observing none, we suggest that the engineering protocols did not adversely impact the cellular and structural integrity of HEK293 cells.

[Part 3: Validation of Engineered HEK293 Cells through Bradford Assay]

Figure 8 (The BSA standard curve was used for quantifying the proteins presented in cell culture media)

We then performed to quantify the proteins presented in cell culture media. Bovine serum albumin (BSA) standard curve was utilized as a reference point to quantify total proteins within our cell culture media. From the trendline equation derived from the BSA standard curve, we established a relationship between the absorbance values obtained from our samples and the corresponding protein concentrations. Using this relationship, we calculated the protein concentrations for each condition tested in our experiment (Figure 8). The results of this quantification revealed the following total protein concentrations (in µg/mL) within the respective cell culture media conditions:

  • Negative control HEK293: 0.81 µg/mL
  • 1:2.5 transfected HEK293: 0.79 µg/mL
  • 1:5 transfected HEK293: 0.75 µg/mL

This is another paramount checkpoint of our research: assessing the impact of our experimental manipulations on protein levels in the cell culture media. Observing negligible differences between the three cell culture media conditions, we suggest that our experimental protocols do not necessarily impair the molecular integrity of the engineered HEK293 cells. This finding, in conjugation with Part 3, demonstrates that our experimental exploitation of the PiggyBac Vector System (and its associated protocols) does not adversely impact HEK293 cells.

[Measurement Protocols]

Bradford assay is a molecular technique utilized for the quantification of protein concentration. The reaction relies on the amino acid composition of target proteins and the dye’s (Coomassie Brilliant Blue G-250) color change in response to protein binding. The dye interacts with positively charged amino acid residues on the surface of proteins, causing the solution to change color. Upon treatment in an acidic solution, the dye binds with proteins to shift the color from brown to blue. The intensity of this color change is directly proportional to the protein concentration in the sample. Key procedures involve the following:

  • Preparation of the BSA standard curve (Figure 8): Spectrophotometric analysis of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 µg/µL BSA solutions.
  • Preparation of sample wells and spectrophotometric absorbance: Addition of Bradford reagent to protein samples.
  • Calculation of protein concentration: Spectrophotometric analysis of reacted protein samples at 595 nm followed by calculations using the BSA standard curve.

It must be noted that all spectrophotometric measurements were recorded in arbitrary units. The least-square regression line of the standard BSA curve is shown below:

  • Y = 0.286 + 0.834*X, R=0.99
  • Absorbance (595 nm) = 0.834 x Total Protein concentration (µg/µL) + 0.286
  • Total Protein concentration (µg/µL) = [Absorbance (595 nm) - 0.286] / 0.834

[Part 4: Validation of Engineered HEK293 Cells through Western Blotting]

Furthermore, to validate that the engineered HEK293 cells, indeed, are capable of synthesizing Wnt3a and Afamin proteins, we performed western blotting. We performed two trials: first with 1 µg of total proteins loaded per well and second with 10 µg of total proteins loaded per well. The first trial, however, was limited with low signal intensity observed for both Afamin and Wnt3a proteins, rendering accurate quantification challenging. Accordingly, we responded by loading 10 µg of total proteins per well to enhance detection sensitivity and obtain solid data.

Figure 9 (Quantification of Afamin and Wnt3a in conditioned media collected. For the first trial, 1 µg of total proteins were loaded per well. For the second trial, 10 µg of total proteins were loaded per well)

Per western blot analysis, we demonstrated that engineered HEK293 cells (transfected with the vector plasmid) exclusively express both Afamin (at approximately 75 kDa) and Wnt3a (at approximately 37.5 kDa) proteins (Figure 8). This validates our ‘Build’ cycle by showcasing the successful integration of our genetic construct into the HEK293 genome. However, owing to the lack of normalization references for both Afamin and Wnt3a, the molar ratio, or relative abundance, of Afamin and Wnt3a proteins is yet to be elucidated and is a ground for future investigation.

Generating Different Builds

Throughout our experiments, we consistently utilized six new Parts we added to the iGEM Registry of Standard Biological Parts. However, for adequate validation and screening procedures, we generated different builds for the following experiments:

  • Part 1: We prepared three plasmid samples (#1, #2, and #3, respectively, Figure 6). Observing proper amplification in only plasmid vector #1–with plasmid vectors #2 and #3 suggesting potential mutation or structural alteration–we utilized plasmid vector #1 for our downstream experiments, ensuring accuracy and precision in our experiments.
  • Part 2: We performed morphological analysis on three experimental samples––the negative control, the 1:2.5 transfected sample, and the 1:5 transfected sample (Figure 7). From the absence of morphological variations, we ascertained that our engineering protocols do not impair the structural integrity of the engineered HEK293 cells.
  • Part 3: We performed Bradford Assay and total protein quantification on three experimental samples–the negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 8). Accordingly, we concluded that our experimental manipulations do not impede host cells’ ability to produce proteins and engage in normal cellular processes (thereby observing the preservation of molecular integrity).
  • Part 4: We performed western blot analysis on three experimental samples–the negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 9). This enabled us to validate bona fide engineered HEK293 cells that contain Afamin and Wnt3a genes.

Screening for Correct Builds

Throughout our ‘Build’ cycle, we extensively screened for correct builds. Primarily, each part of our ‘Build’ cycle was implemented with a corresponding screening measure:

  • Part 1: We performed PCR for three vector plasmid samples. With plasmid vectors #2 and #3 presenting multiple amplified bands, we speculated structural alteration or mutation in the vector plasmids. Accordingly, we responded by utilizing plasmid vector #1–which showed the exact size of the amplified DNA product for both Afamin (250 bp) and Wnt3a (200 bp)--for our downstream experiments (Figure 6). We screened for accurately amplified BII-CMV-AfmW3A vectors upon amplification via E. coli DH5-alpha.
  • Part 2: Through brightfield microscopy, we observed negligible morphological differences between three experimental conditions: the negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 7). We demonstrate that our protocols that involve puromycin treatment and lipofectamine transfection do not adversely impact both the structural and cellular integrity of the engineered HEK293 cells. We screened for the viability and integrity of our engineered cells.
  • Part 4: Through western blot analysis, we observed the production of both Afamin and Wnt3a proteins exclusively in the engineered HEK293 cells (co-transfected with PiggyBac Vector System, Figure 9). This validates the success of our experimental design and approach. This screening measure allowed us to conclude that the HEK293 cells successfully integrated both Wnt3a and Afamin genes–enabling us to proceed with this build for the downstream experiments (as a proof of concept).

Final Version of the Model

Figure 10 (Engineered HEK293 cells producing Afamin and Wnt3a proteins into conditioned media)

We illustrate our system’s final model as above (Figure 10). Detailed descriptions or depictions of our plasmid vectors (or other Parts added to the iGEM Registry of Standard Biological Parts) can be seen in the ‘Design’ section of this page.

Test

Rationale & Objectives

The previous sections focused on the development of the engineered HEK293 cells containing both Afamin and Wnt3a genes. As a proof of concept, this section investigates the effect of Afamin-Wnt3a conditioned media (CM) on bovine myoblast cell (MyoB) proliferation and differentiation. Accordingly, we performed two experiments for this phase: Prestoblue cell proliferation assay and brightfield microscopy.

Test 1: Prestoblue Cell Proliferation Assay

[Experimental Design]

To comprehensively analyze the impact of Afamin-Wnt3a CM on MyoB cell proliferation, we utilized the Prestoblue assay, a highly versatile and pervasive technique to assess cell proliferation with marked precision and accuracy. The assay quantifies cells' metabolic activity, thereby yielding a reliable estimate of cell numbers. Foremost, we established four experimental conditions:

  • Standard DMEM cell culture media without FBS
  • Standard DMEM cell culture media with 10% FBS
  • Standard DMEM cell culture media without FBS but 10% Afamin-Wnt3a CM
  • Standard DMEM cell culture media without FBS but 20% Afamin-Wnt3a CM

We utilized the PrestoBlue assay and conducted incubations at five distinct time points: 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours. The accompanying graph displays the mean and standard deviation (SD) of these measurements (n=2). Furthermore, we assessed statistical significance using the Mann-Whitney test to calculate p-values. All samples were seeded with the sample number of cells (1.25 x 103 cells).

[Measurement Protocols]

Prestoblue assay is a pervasively utilized molecular technique to quantify the metabolic activity of cells as a proxy for cell proliferation. Non-toxic, colorless resazurin, taken up by the living cells, is reduced by mitochondrial enzymes in metabolically active cells. The reduced resazurin, or resorufin, is a highly fluorescent and colorful compound. Accordingly, the fluorescence quantified by absorbance is an indirect measure for the number of metabolically active cells present in the cell culture–an effective reflection of the changes in cell numbers over time, or cell proliferation. The measurement protocols primarily involve the following:

  • Cell Seeding: Seeding of 1x103 MyoB cells to each well and the preparation of standard curve cells by varying the number of cells to 0, 1x103, 2x103, 5x103, 10x103, and 15x103.
  • Preparation of Test Wells and Prestoblue Treatment: Addition of Prestoblue solution at 0, 24, 48,72, and 96 hours of the treatment, followed by an incubation period.
  • Quantification of Cell Proliferation: Absorbance was measured at 570 nm and 600 nm after the incubation period and the calculation of cell viability.
Figure 11 (The number of cells and absorbance measured using the Prestoblue assay shows a strong correlation (R2=0.991))

It must be noted that all spectrophotometric measurements were recorded in arbitrary units. The least-squares regression line, or the standard curve, presents a strong correlation between the number of cells and the absorbance values of the Prestoblue accuracy, validating the accuracy and consistency of our experimental results (R2=0.991, Figure 11). Our standard curve not only showcases the Prestoblue assay as a valid method of quantifying the changes in live cell numbers but also the success of our optimized experimental conditions for the Prestoblue assay. Our least-squares regression line can be illustrated as below:

  • Absorbance (570/600 nm) = 0.1859 x Number of cells (x103) + 0.2943, R=0.99

We quantified the absorbance of cells (or cell viability) at five incubation periods: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr (Figure 12). Accordingly, the number of live cells (x103 cells) was calculated based on the standard curve equation in Figure 11 (Figure 13).

Figure 12 (Prestoblue assay was used to quantify cell proliferation incubation at five incubation times: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr. The measurements indicate the absorbance (570/600 nm) for duplicate experiments)
Figure 13 (The number of live cells (x10^3 cells) was calculated based on the standard curve equation in Figure 11, and absorbance was measured in Figure 12)

[Results and Implications]

Figure 14 (Afamin-Wnt3a CM enhances cell proliferation. Prestoblue assay was used to quantify cell proliferation incubation at five incubation times: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr. The mean and standard deviation (SD) were plotted in the graph (N = 3). Mann-Whitney test was used to calculate the p-value)

The results demonstrate the crucial role of Afamin-Wnt3a CM in inducing MyoB cell proliferation. Notably, we observed an elevated proliferation rate in MyoB cells cultured with 10% Afamin-Wnt3a CM, as shown by an absorbance reading of approximately 2.0 a.u. (p = 0.0295) compared to the FBS-absent sample (Figure 14). These findings articulate the prospect of Afamin-Wnt3a CM in stimulating cell proliferation, with promising implications for cell-based applications and research. We further speculated that increasing the concentration of Afamin-Wnt3a to 20% would result in a more pronounced proliferation increase.

However, our observations indicated that elevating Afamin-Wnt3a to 20% did not substantially boost cell proliferation (~1.3 a.u.). Additionally, it is worth noting that the proliferation rate was lower when supplementing with Afamin-Wnt3a CM compared to 10% FBS (~4.0 a.u.). This suggests that, at present, 10% FBS cannot be replaced by Afamin-Wnt3a CM supplementation, highlighting the unique roles and potential limitations of these factors in cell culture applications. Overall, we successfully iterated the ‘Test’ phase of our engineering cycle by validating the role of Afamin-Wnt3a CM in regulating MyoB cell proliferation. We also underscore the distinct roles and limitations of Afamin-Wnt3a CM in cell culture applications.

Test 2: Brightfield Microscopy

[Experimental Design]

To analyze the extent of MyoB differentiation via analysis of cell density and size and the formation of cell aggregates, we obtained brightfield images of MyoB cells after 96 hours of incubations. We performed the experiment with four total culture media conditions:

Standard DMEM cell culture media without FBS Standard DMEM cell culture media with 10% FBS Standard DMEM cell culture media without FBS but 10% Afamin-Wnt3a CM Standard DMEM cell culture media without FBS but 20% Afamin-Wnt3a CM [Results and Implications]
Figure 15 (Afamin-Wnt3a CM enhances cell proliferation and differentiation. The cell image was taken after 96 hours of incubation of four types of culture media (CM) conditions: without FBS, with 10% FBS, with 10% Afamin-Wnt3a CM, and with 20% Afamin-Wnt3a CM)

We observed the lowest cell density and smaller cell sizes (as compared to other conditions) in the absence of FBS (Figure 15). By contrast, we noted spindle-shaped cells with robust coverage and density in 10% FBS-cultured MyoB cells. Comparably, MyoB cells cultured with 10% Afamin-Wnt3a CM presented similar cell morphology, whereas a ~50% reduction in cell density was observed as compared to the 10% FBS culture. Interestingly, 20% Afamin-Wnt3a CM-cultured MyoB cells exhibited a formation of cell aggregates.

From the analysis of acquired images, we speculated that the 20% Afamin-Wnt3a CM treatment induces the differentiation of MyoB cells (from myoblasts to myocytes and myotubes). The reduction of cell proliferation as compared to the 10% Afamin-Wnt3a CM-cultured MyoB cells can be potentially attributed to the differentiation process. Accordingly, we validated another aspect of our ‘Test’ phase by demonstrating that Afamin-Wnt3a CM induces both cell proliferation and differentiation of MyoB cells; however, the extent of cell proliferation and differentiation likely varies by the concentration of Afamin-Wnt3a CM.

Learn

Through our ‘Build’ and ‘Learn’ phases of the Engineering Cycle, we actively responded to the core principles of engineering in iGEM and Synthetic Biology.

Learn #1: Solve your project’s problems and use synthetic biology tools and/or experimental techniques to generate expected results.

This year, we aimed to propose Afamin-Wnt3a conditioned CM as a potential substitute for FBS, which is limited by ethical concerns, batch-to-batch variability, and potential transmission of pathogens. We effectively addressed our project goals by accomplishing the following:

  • Build: PCR analysis using Afamin and Wnt3a primers to analyze the accurate, precise amplification of the BII-CMV-AfmW3A PiggyBac Vector via E. coli DH5-alpha (Figure 6).
  • Build: Brightfield microscopy to analyze the cell morphology (thus structural integrity) of HEK293 cells co-transfected with the PiggyBac Vector System (Figure 7).
  • Build: Bradford assay using standard BSA curve to analyze the molecular integrity of engineered HEK293 cells by quantifying total protein concentration (Figure 8).
  • Build: Western blot analysis of engineered HEK293 cells to validate the expression and production of Afamin and Wnt3a proteins exclusively in transfected cells (Figure 9).
  • Test: Cell proliferation assay to analyze the effect of Afamin-Wnt3a conditioned CM on proliferation of MyoB cells as compared to FBS (Figures 11~14).
  • Test: Brightfield microscopy to analyze the effect of Afamin-Wnt3a conditioned CM on differentiation of MyoB cells as compared to FBS and previous experiments (Figure 15).

Overall, we performed six rounds of experimentation (involving unique synthetic biology tools) to generate the expected results. We demonstrated that the engineered HEK293 cells co-transfected with the PiggyBac Vector System inserted Afamin and Wnt3a genes and robustly expressed them. Furthermore, we confirmed that Afamin-Wnt3a conditioned CM induced MyoB proliferation and differentiation--while its extent varies by the concentration. This validates the potential of Afamin-Wnt3a-conditioned CM as a substitute for FBS.

Learn #2: Think about and document what changes in design you would make for the next iteration(s) of the cycle.

While our first iteration of the Engineering Cycle was fruitful, we were limited by the following:

  • Inability to elucidate the relative abundance of Afamin and Wnt3a proteins
  • Lacking a clear-cut explanation of the relationship between MyoB proliferation and differentiation as evinced by Prestoblue assay and brightfield microscopy

To quantify the relative abundance of Afamin and Wnt3a proteins, we must derive a normalization standard for both Afamin and Wnt3a proteins, which is currently lacking in the literature. We highlight the potential of developing novel measurement standards (or new Parts to characterize/quantify Afamin and Wnt3a proteins) to accomplish this gap. This, indeed, will enable us to further understand the distinct roles and limitations of Afamin-Wnt3a conditioned CM in cell culture applications.

Second, to offer a clear-cut explanation of the relationship between MyoB proliferation and differentiation, another round of experimentation/investigation must be implemented. This may involve molecular analysis (such as PCR or western blot analysis) of differentiation-related factors, such as MYOD and MYF5, under varying concentrations of Afamin-Wnt3a conditioned CM. This, when conjugated in relation to cell proliferation assay, can potentially provide a categorical effect that the manipulation of Afamin-Wnt3a conditioned CM’s concentration wields.

Learn #3: Design and build a new Part, measure its performance, document whether it worked or not, and propose how the results would inform the next design or steps.

We added six new Parts to the iGEM Registry of Standard of Biological Parts. We accurately designed six rounds of experiments--spanning one full iteration of the Engineering Cycle--to validate the performance and functionality of the optimized PiggyBac Vector System. The limitations of our experiments attest to the next steps and designs we may take.