Contribution Overview

This year, our team established a useful contribution for future iGEM teams by adding six new parts to the iGEM Registry of Standard Biological Parts. Our Parts combine to serve three foremost purposes: (1) amplification of BII-CMV vectors via E. coli DH5-alpha strain; (2) lipofectamine-mediated co-transfection of PiggyBac Vector System (BII-CMV-AfmW3A and Super PiggyBac Transposase Expression Vector) to establish HEK293 cells containing Afamin and Wnt3a genes; and (3) expression of Afamin and Wnt3a proteins via HEK293 cells to develop conditioned media that can reliably substitute fetal bovine serum (FBS) in cultured meat production. The integration of our Parts can be further viewed on our Parts Collection page.

For each Part we characterized, we performed a brief literature review and analyzed it within our experimental context. While the literature review does not necessarily pertain to or align with our team’s experimental investigation, it offers foundational insights into and key background knowledge should future iGEM teams consider these Parts for usage. Furthermore, our experimental analysis offers a thorough, extensive guideline to how each Part was utilized in our experimental context--and what results were obtained to garner which conclusions; we speculate these pieces of information will usefully guide future iGEM teams in navigating their design, engineering, and validation processes. Our newly added parts are as below:

• BII-CMV-AfmW3A Vector (Part: BBa K4869000)
• Super PiggyBac Transposase Expression Vector (Part: BBa K4869001)
• Engineered HEK293 cells containing afamin and wnt3a genes inside the genome (Part: BBa K4869003)
• Afamin gene (Part: BBa K4869004)
• Wnt3a gene (Part: BBa K4869005)
• Terminal inverted repeat sequence (Part: BBa K4869006)

Overall, the combination of newly added Parts articulates the potential usage of the PiggyBac Vector System in multiple bioengineering applications. The System enables stable, long-term integration and expression of multiple genes of interest into various mammalian cell lines. This year, we engineered HEK293 cells that contain Wnt3a and Afamin genes by exploiting this System. The experimental analysis offered in each Part’s characterization validates both the success and efficiency of our approach--and stepwise manual for future iGEM teams to customize the System for their research purposes. In particular, we highlight the genetic construct utilized in the BII-CMV-AfmW3A Vector and the co-transfection of BII-CMV-AfmW3A Vector and Super PiggyBac Transposase Expression Vector into HEK293 cell lines.

1. BII-CMV-AfmW3A Vector (Part: BBa K4869000)

General Information & Literature Review

This involved employing PCR and gel electrophoresis techniques to assess the presence of the proteins in the BII-CMV-AfmW3A vector with a circular DNA form consisting of 10,434 base pairs. Each gene within the vector, including CMV promoter, Afamin, T2A sequence, Wnt3a, puromycin resistance gene, and ampicillin resistance gene, plays a distinct role in producing Afamin-Wnt3a conditioned media.

Application in Our Project

This project aimed to generate Afamin-Wnt3a-producing cells to obtain an FBS-free growth medium for cultivating bovine muscle cells. We utilized a piggyBac vector (BII-CMV-AfmW3A) to express Afamin and Wnt3a (as shown in Figure 1) and the Super piggyBac transposase expression vector to transfect human embryonic kidney 293 cells (HEK293).

The BII-CMV-AfmW3A vector is known to be effective when inserting foreign DNA (Afamin-Wnt3a) into the host genome (HEK293). The BII-CMV-AfmW3A vector is made up of a circular DNA consisting of 10,434 base pairs. The vector contains various genes such as CMV promoter, T2A sequence, puromycin resistance gene, Afamin, and Wnt3a gene. Our study mainly focused on two genes, which are the Afamin and Wnt3a genes, which are included in the BII-CMV-AfmW3A vector.

Our goal was to transfect the two genes Afamin and Wnt3a into HEK293 cells and insert Afamin and Wnt3a into the genome of the cell. 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, as an alternative to the fetal bovine serum (FBS). Therefore, the piggyBac vector was used as a mechanism to easily transfer the Afamin and Wnt3a gene into the HEK293 cells. This vector will later be amplified after being transformed into DH5alpha E.coli.

References

Loidolt S., Establishment of Afamin-Wnt3a producer cells to generate serum-free growth medium for canine intestinal organoid culture. https://phaidra.vetmeduni.ac.at/open/o:869.

2. Super PiggyBac Transposase Expression Vector (Part: BBa K4869001)

General Information & Literature Review

The PiggyBac (PB) transposon is a type of mobile genetic element capable of transferring genetic material between vectors and chromosomes in a highly efficient manner via a "cut and paste" mechanism. The PB transposase identifies specific inverted terminal repeat sequences (ITRs) located at both ends of the transposon vector during the transposition process and moves the contents from their original sites, integrating them efficiently into TTAA chromosomal sites. This piggyBac transposon system makes it easy to transfer genes of interest between the two ITRs in the PB vector into target genomes.

The super piggyBac transposase expression vector also has a circular shape and consists of 6,964 base pairs, making it relatively smaller than the BII-CMV-AfmW3A vector. This vector contains CMV promoter, SV40 late 16s intron, Super PiggyBac Transposase, and SV40 poly-A. PiggyBac is a transposon, a DNA sequence that can move around within the genome of a cell. It has been adapted for use in genetic engineering, allowing the insertion, deletion, or movement of genes within the genome of various organisms. It is a hyperactive version of the enzyme that efficiently integrates any-sized DNA insert into the genome. The Super PiggyBac transposase expression vector features a strong CMV promoter to provide robust transposase expression. The PiggyBac transposase coding sequence has been optimized for high expression, stability, and integration activity in mammalian cells.

Application in Our Project

Super piggyBac transposase expression vector is a plasmid vector that was used to supplement the process of BII-CMV-AfmW3A vector genes successfully getting inserted into the host genome (HEK293). The Super piggyBac system is a powerful tool for introducing and stably integrating genetic material into host cells, ensuring long-term and stable expression of the desired genes. With the BII-CMV-AfmW3A vector, the super piggyBac transposase expression vector was used in vector transfection, which is a process of artificially putting external DNA into the target cell. This vector transfection facilitated the engineering process of HEK293 cells to produce the Afamin-Wnt3a protein complex by the insertion of Afamin and Wnt3a genes into the host cell.

References

3. Engineered HEK293 cells containing afamin and wnt3a genes inside the genome
(Part: BBa K4869003)

Application in Our Project

Our first goal is to establish Afamin-Wnt3a producer cells in vitro. We will do this by transfecting cells with a plasmid containing the Afamin-Wnt3a gene. Once the cells successfully produce Afamin-Wnt3a protein, we will use them to generate conditioned media for our downstream experiments.

Transfection is the process of introducing foreign DNA or RNA into cells. In this case, we want to introduce a plasmid containing the Afamin-Wnt3a gene into HEK293 cells. Once the plasmid is inside the cell, it can be transcribed and translated into Afamin-Wnt3a protein. This plasmid can be transfected into HEK293 cells, which are a type of human embryonic kidney cell line that is commonly used in scientific research.

When the Afamin-Wnt3a gene is expressed in these cells, it leads to the production of the Afamin-Wnt3a protein. One way to introduce the Afamin-Wnt3a Plasmid into HEK293 cells is through the Super piggyBac Transposon vector. Super piggyBac Transposon vector contains the PiggyBac transposase gene, an enzyme that can integrate the Afamin-Wnt3a Plasmid into the genome of the HEK293 cells. Once integrated, the Afamin-Wnt3a gene sequence becomes a part of the cell's DNA and is passed on to daughter cells during cell division.

By using the Super piggyBac Transposon vector to generate Afamin-Wnt3a producer cells, we can produce large quantities of Afamin-Wnt3a protein for use in downstream experiments. With a combination of those following genomes, the plasmid containing the Afamin-Wnt3a gene must be transfected to the genome of HEK293 cells to establish Afamin-Wnt3a producer cells. HEK293 cells are also known as human embryonic kidney cells, which are robust and fast-growing and, therefore, have been frequently used for receptor signaling, cancer research, and large-scale protein production.

To successfully integrate the foreign genes into the host genome, one strategy is to utilize the Super piggyBac Transposon vector. Super piggyBac transposase gene inside the transposon vector is the enzyme playing the crucial role of transfecting the plasmid into the HEK293 cells genome. Once transfected, the Afamin-Wnt3a gene will be integrated within the HEK293 cells’ DNA and passed on to other cells during cell division. Consequently, Afamin-Wnt3a producer cells may be generated utilizing the Super piggyBac Transposon vector.

We utilized the engineered HEK293 cells by designing the cells, using Super piggyBac transposase expression vector and BII-CMV-AfmW3A vector, to produce Afamin Wnt3a protein complex enriched conditioned media. To determine the optimal conditions for the transfection process, we conducted experiments with two different plasmid vector molar concentration ratios between the BII-CMV-AfmW3A vector and Super PiggyBac Transposase Expression Vector. After the Afamin Wnt3a protein complex, which is initially made in the cytosol of the cell, is secreted into the cell media by the HEK293 cell, we collected the enriched conditioned media, which is a replacement for FBS.

References

4. Afamin gene (Part: BBa K4869004)

General Information & Literature Review

Mus mucus (mouse) Afamin gene was used in this study (1824 bp). Afamin, otherwise named alpha-albumin, alpha-1D-glycoprotein, vitamin E-binding protein, is a glycoprotein that is first synthesized in the liver of an organism and then secreted into the bloodstream. This gene is a member of the albumin gene family, comprising four localized genes to chromosome 4 in tandem arrangement. It also belongs to the albumin protein family while structurally similar to serum albumin.

Application in Our Project

Afamin proteins serve various, integral roles in human physiology. The first major function of afamin is being capable of combining and transporting vitamin E in the blood across the blood-brain barrier. Vitamin E, in this scenario, is a crucial antioxidant that acts as a protection layer from possible oxidative damage caused by free radicals; this also facilitates the immune system. Maintaining vitamin E levels ensures tissue delivery to multiple places in the body. By this function, the combination of Afamin and Wnt3a can proliferate bovine cells that contribute to the development of lab-cultured meat, as shown from the visual of cell proliferation and the subsequent graph of this data (Figures 4 and 6).

The previous research showed that Afamin forms a complex with the Wnt3a protein (Figure 5). This complex helps to stabilize the Wnt3a protein and prevents its self-aggregation in the conditioned media. Therefore, as a result, the coexpression of Afamin and Wnt3a would enable the production of stable Wnt3a proteins, which enhances the Wnt3a-dependent cell proliferation signaling in bovine muscle cells.

Further, Afamin serves other physiological processes, such as regulating inflammation, modulating the immune response, and affecting insulin sensitivity. Additionally, recent studies have discovered that afamin can be used as a biomarker for ovarian and other types of cancer. The clinical-grade analysis and study of Afamin proteins lead to the conclusion that Afamin assays meet the laboratory medicine thus, comparative proteomics has identified it as a potential biomarker in the human body.

Moreover, Afamin is also related to the prevalence and development of metabolic syndrome. This was discovered through the basic knowledge that transgenic mouse data had a strong association with the aforementioned function. In our project, Afamin is integral in proliferating bovine cells when functioning with Wnt3a. As the combination of these two proteins induces signaling of various cellular systems, including intestinal stem cell growth, these two proteins create an ideal environment for bovine muscle growth and allow for bovine cell proliferation.

References

5. Wnt3a gene (Part: BBa K4869005)

General Information & Literature Review

Homo sapiens (human) Wnt3a gene was used in this study (1059 bp). Wnt3a is a member of the Wnt family of secreted signaling proteins that play a crucial role in cell proliferation and other cellular processes. Wnt3a activates the canonical Wnt signaling pathway, which regulates the transcription of various genes involved in cell proliferation, differentiation, and survival. Wnt3a has been shown to play a critical role in the proliferation of stem cells, as well as the maintenance of adult tissues such as the intestine, skin, and hair follicles. Studies have also suggested that aberrant Wnt3a signaling can contribute to the development and progression of various types of cancer, as dysregulated Wnt signaling can promote uncontrolled cell proliferation and tumor growth. Wnt3a is a key ligand of the canonical Wnt/β-catenin pathway and supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased livers.

The in-depth exploration of the Wnt3a gene sequencing data may be facilitated through rigorous examination of online databases, notably GenBank, Ensembl, and the UCSC Genome Browser. These databases not only proffer sequence information but also offer annotations that are pivotal for comprehending the genomic organization of the gene, incorporating both exons (coding segments) and introns (non-coding segments).

Wnt3a, in its role as a ligand, engages with Frizzled receptors on cellular surfaces, thereby precipitating a multitude of intracellular signaling pathways, notably the canonical β-catenin-dependent pathway. This mechanism proves fundamental in the regulation of cellular fate and in bolstering embryonic development. Moreover, aberrations in the expression or functionality of Wnt3a have been associated with a diverse array of pathologies, including cancers, due to its salient role in regulating cellular proliferation and differentiation. Therefore, the utilization of scientific databases and literature, which go through regular updates and peer review, is paramount to secure accurate information regarding the sequence and attributes of the Wnt3a gene.

Application in Our Project

Experimentally, the Wnt3a gene has been substantiated to enhance the proliferation of bovine muscle cells. By incorporating Wnt3a in conditioned media, there is potential to amplify the proliferation and differentiation of bovine muscle cells, thereby offering a pathway toward a more economical and sustainable methodology for beef production. Furthermore, Wnt signaling has the aptitude to stimulate the proliferation of muscle stem cells, known as satellite cells, which are indispensable in the production of cultured meat. The activation of the Wnt pathway can induce satellite cells to divide and differentiate into mature muscle cells, which can subsequently be harvested and processed into meat products while upholding principles of sustainability and economic viability.

References

6. Terminal inverted repeat sequence (Part: BBa K4869006)

General Information & Literature Review

Terminal inverted repeat (TIR) sequences are essential for the transposition of most transposons. They are distinct DNA structures found at the ends of certain transposable elements (TEs). TIR sequences are substantially similar and are made of two sequences that are inverted complements of each other, meaning that the complementary strand would read right to left in one strand of the TIR was read from left to right. Typically, the inverted repetitions range in length from a few to several hundred base pairs.

For the transposition of many different forms of transposable elements, notably DNA transposons, TIR sequences are crucial. The transposase enzyme, which is in charge of slicing and pasting the transposon into a new position in the genome, uses them as recognition sites. The excision and integration of the transposon are facilitated by the transposase enzyme, which identifies and binds to the TIR sequences.

Application in Our Project

In experimental contexts, TIR sequences are essential in utilizing the maneuverability of DNA transposons and certain retrotransposons, facilitating precise genetic insertions via a "cut and paste" methodology. Particularly, the piggyBac transposon system, utilizing TIR sequences, enables scientists to strategically insert genes into a host genome, such as HEK293 genomic DNA, by employing a transposase enzyme. This system proves invaluable in gene therapy and genetic engineering experiments by allowing for the meticulous and targeted insertion of therapeutic or desired genes, thereby enhancing the efficiency and accuracy of genetic manipulations and interventions within research and biotechnological applications.

References

Reflection

Cumulatively, we contributed to the work of future iGEM teams by registering six new Parts to the iGEM Registry of Standard Biological Parts. Each Part was accompanied by a thorough literature review and experimental guideline/analysis based on our investigations. We, in particular, highlight the potency of the PiggyBac Vector System in future iGEM endeavors, a powerful tool to integrate multiple genes of interest into the host genome and mimic the polycistronic mRNA-like expression of multiple genes by the host cells.

Our team has successfully validated this approach by co-transfecting BII-CMV-AfmW3A and Super PiggyBac Transposase Expression Vectors into HEK293 cells. We concluded that the engineered HEK293 cells produce Afamin and Wnt3a proteins, critical supplements for our final conditioned media. We also suggest the significance of terminal inverted repeat sequence (ITR) in the overall genomic integration process. Overall, this method could be customized to various research endeavors for speedy, efficient engineering of various mammalian cell lines--and the production of complex eukaryotic proteins.

Parts Collection

Primary Objective

Our collection of Parts serves three primary purposes: (1) amplification of BII-CMV-AfmW3A vectors via E. coli DH5-alpha; (2) co-transfection of amplified BII-CMV-AfmW3A and Super PiggyBac Transposase Expression Vector to engineer HEK293 cells containing Afamin and Wnt3a genes; and (3) production of Afamin and Wnt3a via HEK293 cells to develop conditioned media as a potential substitute for fetal bovine serum (FBS) in cultured meat synthesis.

This year’s Parts Collection, in fact, primarily relied on our endeavors in Human Practices. Various stakeholders suggested improvements in production cost and speed (i.e., efficiency)--and the ultimate quality of the cultured meat. Our Parts Collection responds to synthetic biology-based, experimental solutions to the status quo and voices cardinally incorporated from diverse stakeholders.

Parts Collection

For our 2023 iGEM project, we utilized the following collection of parts:

BII-CMV-AfmW3A Vector (Part: BBa K4869000)
Super PiggyBac Transposase Expression Vector (Part: BBa K4869001)
Engineered HEK293 cells containing afamin and wnt3a genes inside the genome (Part: BBa K4869003)
Afamin gene (Part: BBa K4869004)
Wnt3a gene (Part: BBa K4869005)
Terminal inverted repeat sequence (Part: BBa K4869006)

Parts as a Whole

Our collection of Parts functions as a ‘system’: PiggyBac Transposon System. The System enables speedy, efficient transgenesis with no limits on cargo size. Conventionally, the system comprises co-transfection of two (or more, occasionally) vectors: the PiggyBac vector and the Super PiggyBac Transposase Expression Vector. Super PiggyBac Transposase Expression Vector synthesizes Super PiggyBac Transposase, which recognizes PiggyBac Vector’s inverted terminal repeats (ITRs). Subsequently, ITRs and intervening DNA are effectively integrated into the host genome at TTAA sites, engineering the host cells.

The first Part of this System is the BII-CMV-AfmW3A Vector (Part: BBa K4869000), which its genomic sequence can be found in Figure 1. Primarily, the Vector contains ITR sequences at both 5’ and 3’ ends (Part: BBa K4869006). The ITR sequences are intervened by Afamin (Part: BBa K4869004) and Wnt3a (Part: BBa K4869005) genes, the primary supplements of our conditioned media. The Vector also contains several elements critical to the System’s success, which can be found below:

CMV Promoter: Cytomegalovirus promoter, or CMV promoter in short, is a pervasively utilized promoter for various bioengineering purposes. It wields constitutive activity and robustness in mammalian cell lines--and thus initiates transcription effectively.

2A: The 2A peptide cleavage system enables co-expression of Afamin and Wnt3a from a single mRNA transcript. Accordingly, the system was placed between Afamin and Wnt3a genes to effectively co-express them.

IRES: Internal ribosome entry site (IRES) sequence empowers the co-expression of Afamin and Wnt3a on a single mRNA transcript by initiating cap-independent translation and fusion of the co-expressed genes.

PuromycinR: The Puromycin resistance gene is a measure for negative selection of successfully transformed HEK293 cells by eliminating unsuccessfully transformed cells.

WRPE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WRPE) sequence induces expression of Wnt3a and Afamin by enhancing mRNA stability and cytoplasmic accumulation.

SV polyA: SV40 polyadenylation sequence (SV polyA) effectively terminates the transcription of intervening DNA sequence and thereby ensures accurate, precise mRNA processing and efficient translation of Afamin and Wnt3a proteins.

The second part of this system is the Super PiggyBac Transposase Expression Vector (Part: BBa K4869001). The Vector, as previously indicated, synthesizes Super PiggyBac Transposase, which in turn integrates the sequence depicted in Figure 1 (intervened by the ITR sequences) into the host genome at TTAA sites. Two vectors are co-transfected into HEK293 cells via lipofectamine (Figure 2). This leads to the production of engineered HEK293 cells that contain Afamin and Wnt3a genes inside their genome (Part: BBa K4869003).

Overall, our system in this year’s iGEM project enabled speedy, efficient, and accurate production of Wnt3a and Afamin-enriched media. This, indeed, appropriately responds to both (1) key bioengineering protocols and principles underlying Synthetic Biology and (2) feedback voiced by various stakeholders we interacted with.

Validation of Parts Collection

The foremost checkpoint in our validation of Parts Collection is the vitality of engineered HEK293 cells. Indeed, it is principally plausible that the process of genomic integration--lipofectamine-induced co-transfection and puromycin treatment--hampers the molecular and cellular viability of engineered HEK293 cells.

We observed negligible differences in cellular morphology for HEK293 cells subject to varying experimental treatments: negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 3). A comparable cellular morphology suggests both the overall health and stability of the engineered HEK293 cells. Furthermore, the results demonstrate that our collection of Parts does not adversely affect the molecular and structural integrity of the modified cells.

Furthermore, we utilized the bovine serum albumin (BSA) standard curve--a primary point of reference--to quantify the proteins within cultured media via Bradford assay (Figure 4). By obtaining the relationship between absorbance values of the samples and corresponding protein concentrations, we quantified protein concentrations for each condition as below:

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

Here, we also find a negligible difference in protein concentrations. This, again, attests to the viability, stability, and vitality of the engineered HEK293 cells, thereby enabling us to demonstrate the limited effect of experimental manipulations on the engineered cells.

As a final measure to conclusively validate our collection of Parts, we performed a western blot to detect Afamin and Wnt3a proteins in conditioned media. We present two experiments--loading 1 µg total protein per well and 10 µg total protein per well, respectively. Acknowledging the challenges of accurate protein quantification in the first experiment (as evidenced by the low signal intensity), we subsequently loaded 10 µg total protein per well.

The analysis of protein via western blot analysis demonstrated exclusive expression of Wnt3a (≈37.5 kDa) and Afamin (≈75 kDa) proteins in transfected cell lines (Figure 5). While we experimentally were limited in quantifying the molar ratio of Afamin and Wnt 3a proteins (due to a lack of references for normalization), we successfully engineered HEK293 cells that effectively produce Wnt3a and Afamin genes.

Reflection

Overall, we validated our collection of Parts through three separate experiments. First, the structural integrity of engineered HEK293 cells (Figure 3); second, the molecular integrity of engineered HEK293 cells (Figure 4); and third, the successful production of Afamin and Wnt3a proteins by the engineered HEK293 cells (Figure 5).