Contribution

Contribution (Parts)

1. Overview

<Overview>

Our team contributed both newly domesticated parts and improved documentation for existing Registry entries. We created RFC[10]/RFC[1000]-compatible versions of the human EF1α promoter, NRAS wild-type, NRAS G12D, mCherry, and the puromycin resistance gene (PuroR) by removing internal restriction sites through synonymous substitutions while preserving their amino acid sequences and functions. These standardized versions provide ready-to-use building blocks for mammalian expression systems. We also utilized existing Registry resources such as the Kozak consensus sequence, T2A/P2A peptides, luciferin-related parts, and polyA signals (BGH, SV40 late), adding detailed notes to the Documentation section to guide future teams on how these elements can be applied in construct design.

To demonstrate the practical application of these parts, we assembled two composite constructs: EF1α promoter – hNRAS^WT – mCherry – PuroR and EF1α promoter – hNRAS^G12D – mCherry – PuroR. These modular expression cassettes provide reference systems for studying NRAS function, oncogenic mutations, and their impact on cell proliferation and signaling, while also offering convenient visualization and stable cell selection. By sharing both domesticated parts and composite examples, our contribution expands the Registry with resources that are directly usable in mammalian systems and adaptable for diverse synthetic biology designs.

2. Human EF1α promoter — RFC[10]/RFC[1000]-compatible (modified) (BBa_25GHPWMW)registry.igem.org/parts/bba-25ghpwmw

Sequence information:
GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAATACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTAGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAAGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA

<Registry Contribution>

We contributed a modified version of the human EF1α promoter that is fully RFC[10]/RFC[1000]-compatible. This domesticated sequence was generated by removing internal restriction sites through synonymous substitutions, preserving its strong and stable transcriptional activity in mammalian cells. By providing this standardized promoter, future iGEM teams can readily incorporate EF1α into plasmid design or mammalian expression constructs, taking advantage of its broad utility without compatibility issues.

<General Information & Literature Review>

The EF1A promoter originates from the human elongation factor-1 alpha (EEF1A1) gene, which encodes a highly expressed translation factor in mammalian cells.

Promoter Characteristics:

  • Constitutive Expression: Unlike inducible promoters (e.g., tetracycline- or CMV-inducible systems), the EF1α promoter drives continuous transcription of downstream genes without requiring an external inducer.
  • Stable Activity Across Cell Types<: EF1α is ubiquitously expressed in mammalian cells, including fibroblasts, stem cells, and tumor cells, making it suitable for diverse applications.
  • Reduced Silencing compared to CMV: While the CMV promoter is strong, it is prone to epigenetic silencing in long-term culture. EF1α is more resistant to silencing, ensuring persistent gene expression.
  • Eukaryotic Origin: As a promoter derived from a housekeeping gene in eukaryotes, EF1α functions efficiently in mammalian chassis such as NIH3T3 cells.

Promoter selection rationale:

  • Avoids dependence on chemical inducers, ensuring lower cost and simpler handling compared to inducible systems.
  • Provides robust, stable, and long-term expression, which is critical for comparing phenotypic effects of NRAS^WT versus NRAS^G12D in NIH3T3 cells.
  • Widely validated in literature: EF1α has been successfully used in gene therapy, stem cell research, and cancer models, demonstrating reliability and broad applicability.
  • Safety: Because it is derived from a mammalian housekeeping gene and not from an antibiotic resistance marker, it is considered safe and non-immunogenic in cell culture contexts.

<Application in Our Project>

In our iGEM project, the EF1α promoter is employed to drive constitutive expression of the NRAS^WT and NRAS^G12D constructs in NIH3T3 cells.

  • Goal: Provide stable and continuous production of NRAS transcripts and proteins in order to directly compare phenotypic effects between the wild-type and mutant alleles without the influence of external inducers.
  • Design Considerations:
    • ○ The EF1α promoter ensures long-term, consistent expression in mammalian cells, reducing variability caused by promoter silencing or fluctuating culture conditions.
    • ○ Constitutive expression allows differences in cell growth, viability, and migration to be attributed primarily to the NRAS mutation rather than variability in induction protocols.
    • ○ By combining EF1α with fluorescent and luminescent reporters, our system supports both qualitative monitoring and quantitative measurement in 2D and 3D culture environments.
References

Wang X. et al. (2017). The EF-1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number.Gene Therapy, 24(9): 671–679.

  1. [1] Kozíšek T. et al. (2021).Comparison of promoter, DNA vector, and cationic carrier on transfection efficiency and transgene expression.International Journal of Molecular Sciences, 22(16): 8929.
  2. [2] Qin J.Y. et al. (2010).Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter in Lentiviral Vectors.PLoS ONE, 5(5): e10611.
  3. [3] Cabrera A. et al. (2022). Transgene silencing in mammalian cell engineering.Frontiers in Bioengineering and Biotechnology, 10: 988085.

3. Kozak(BBa_K5073037)registry.igem.org/parts/bba-k5073037

Sequence information:
GCCACC

<Registry Contribution>

We contributed to the Registry by utilizing the existing information on the Kozak consensus sequence and incorporating it into our mammalian expression cassette design. In addition, we documented the application of this sequence within our composite design (EF1α–NRAS–mCherry–PuroR) and added detailed notes in the Documentation sections. This provides future teams with both a ready-to-use sequence and practical context for its implementation.

<General Information & Literature Review>

The Kozak consensus sequence originates from systematic analysis of eukaryotic mRNAs by Marilyn Kozak in the 1980s. It represents a conserved nucleotide motif that enhances the efficiency of translation initiation at the AUG start codon.

Characteristics:

  • Translation Efficiency: The Kozak sequence facilitates accurate ribosomal recognition of the start codon, thereby increasing translation initiation efficiency.
  • Universality in Mammalian Cells: It has been validated across a wide range of mammalian expression systems, making it a standard feature in eukaryotic expression vectors.
  • Non-coding Element: Unlike promoters or ORFs, it does not encode a protein but acts purely at the level of translational control.
  • Safety: As a short, non-coding regulatory element derived from natural mammalian mRNAs, it poses no biosafety concerns.

Design Rationale:

  • Ensures that downstream ORFs, such as NRAS^WT or NRAS^G12D, are efficiently translated.
  • Minimizes variability in protein expression that could otherwise confound comparison between wild-type and mutant NRAS constructs.
  • Serves as a universal translational enhancer that avoids reliance on complex 5’ UTR structures.

<Application in Our Project>

In our iGEM project, the Kozak consensus sequence was placed immediately upstream of the NRAS^WT and NRAS^G12D start codons.

Goal: Guarantee robust and reproducible initiation of NRAS translation in NIH3T3 cells, ensuring that phenotypic differences observed are due to the mutation itself rather than inconsistencies in translation initiation.

Design Considerations:

  • By including the Kozak sequence, we standardize translational efficiency between NRAS^WT and NRAS^G12D constructs.
  • This design reduces experimental variability and allows reliable downstream assays (cell growth, viability, migration, drug response) to reflect the genetic difference rather than regulatory noise.
  • The Kozak sequence complements the EF1α promoter, creating a strong and stable transcription-translation initiation module.
References
  1. [1] Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell, 44(2), 283–292.
  2. [2] Kozak, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research, 15(20), 8125–8148.
  3. [3] Hernandez, G. et al. (2019). The importance of the Kozak sequence in translation initiation in eukaryotes. EMBO Reports, 20(10), e48235.
  4. [4] Mishra, R. et al. (2019). Effect of Kozak sequence variants on gene expression in mammalian cells. Molecular Biotechnology, 61(6), 421–431.

4. hNRAS gene

- Wild type(BBa_251QYRB4)https://registry.igem.org/parts/bba-251qyrb4
- G12D mutation type(BBa_252RMATF)https://registry.igem.org/parts/bba-252rmatf

Sequence information:

1. Wild type(WT) gene sequence

ATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGT*GGTGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGATTCTTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACAAGAAGAGTACAGTGCCATGAGAAACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATAGCAAGTCATTTGCGGATATTAACCTCTACAGGGAGCAGATTAAGCGAGTAAAAGACTCGGATGATGTACCTATGGTGCTAGTGGGAAACAAGTGTGATTTGCCAACAAGGACAGTTGATACAAAACAAGCCCACGAACTGGCCAAGAGTTACGGGATTCCATTCATTGAAACCTCAGCCAAGACCAGACAGGGTGTTGAAGATGCTTTTTACACACTGGTAAGAGAAATACGCCAGTACCGAATGAAAAAACTCAACAGCAGTGATGATGGGACTCAGGGTTGTATGGGATTGCCATGTGTGGTGATG

2. G12D mutation type gene sequence

ATGACAGAGTACAAACTGGTGGTGGTGGGGGCCGAC*GGGGTGGGGAAAAGCGCCCTGACCATTCAGCTGATCCAGAATCACTTTGTGGACGAGTACGACCCAACCATCGAGGACAGCTATAGAAAACAGGTCGTGATCGATGGTGAAACCTGTCTGCTGGACATCCTGGACACTGCAAGCCAGGAGGAGTACTCTGCCATGAGAAACCAGTACATGAGGACCGGCGAGGGATTTCTGTGCGTGTTTGCTATCAACAACTCTAAGAGTTTCGCCGATATCAATCTGTACAGAGAGCAGATCAAAAGGGTGAAGGACAGCGACGACGTGCCCATGGTGCTGGTGGGCAATAAGTGCGACCTGCCAACCCGGACAGTGGATACCAAGCAGGCCCACGAACTGGCCAAGAGTTACGGGATCCCTTTCATCGAAACCAGCGCCAAGACCAGACAGGGCGTGGAGGACGCCTTCTACACCCTGGTGAGGGAGATTCGGCAGTACCGCATGAAGAAGCTGAATAGCTCCGACGACGGAACACAGGGCTGCATGGGCCTGCCCTGTGTGGTGATG

<Registry Contribution>

1. Wild type(WT)
We contributed a domesticated version of the human NRAS wild-type coding sequence, modified to be RFC[10]/RFC[1000]-compatible through synonymous substitutions. This standardized sequence preserves the original amino acid content while removing restriction sites. By providing an accessible NRAS wild-type part, future teams can use it as a baseline to investigate how NRAS signaling affects cell proliferation and as a reference point for studying oncogenic variants in cancer biology.

2. G12D mutation type(G12D)
We contributed a domesticated version of the human NRAS G12D oncogenic variant coding sequence, engineered to be RFC[10]/RFC[1000]-compatible without altering its amino acid content. This standardized part enables future teams to study the functional consequences of oncogenic mutations, including how NRAS G12D drives abnormal proliferation and cancer-associated phenotypes. It provides a ready-to-use platform for exploring the genetic basis of tumor progression and therapeutic response.

<General Information & Literature Review>

The NRAS gene encodes a small GTP-binding protein that functions as a molecular switch in signal transduction pathways. It cycles between an inactive GDP-bound state and an active GTP-bound state, regulating key downstream pathways such as MAPK/ERK and PI3K/AKT.

Characteristics:

  • Wild-Type Function: NRAS^WT plays a normal role in cell proliferation, differentiation, and survival. Its activity is tightly controlled by intrinsic GTPase activity and regulatory proteins such as GEFs and GAPs.
  • G12D Variant (hNRAS^G12D): Point mutations at codons 12 (GGT → GAC) locks NRAS in its active GTP-bound form. This results in constitutive signaling, uncontrolled proliferation, and resistance to apoptosis. And promoting malignancy and therapy resistance in hematologic cancers.
  • Biomedical Relevance: NRAS mutations occur in ~20–30% of melanomas and ~15% of AML cases. This makes NRAS a clinically significant model gene for studying oncogenic signaling.
  • Codon Optimization: Both hNRAS^WT and hNRAS^G12D coding sequences were codon-optimized for mammalian expression. As a result, the optimized sequences differ not only at codon 12 but also in synonymous substitutions throughout the ORF.

Design Rationale:

  • Codon optimization ensures robust protein translation in mammalian systems, minimizing expression variability.
  • Comparing NRAS^WT and NRAS^G12D allows a clear genotype-to-phenotype link, making the mutation effect observable in growth, viability, and migration assays.
  • Including both WT and mutant alleles in otherwise identical constructs ensures that phenotypic differences arise solely from the mutation.

<Application in Our Project>

In our iGEM project, hNRAS^WT and hNRAS^G12D coding sequences were cloned into expression vectors under the EF1α promoter, with a Kozak consensus sequence upstream.

  • Goal: Establish stable overexpression of both wild-type and mutant NRAS in NIH3T3 cells, enabling direct assessment of the oncogenic effects of the G12D mutation.
  • Design Considerations:
    • Codon optimization was applied to maximize expression efficiency, but this required designing specific primers for each construct.
    • The codon 12 mutation site was explicitly included in primer design to validate the presence of the G12D substitution.
    • Reporter genes (Luc2, mCherry) were linked via T2A sequences, allowing phenotypic changes to be tracked in real time.
References
  1. [1] Bos, J.L. (1989). ras oncogenes in human cancer: a review. Cancer Res. 49(17): 4682–4689.
  2. [2] Ward, A.F. et al. (2012). RAS oncogenes: signaling, plasticity, and drug resistance. Cell, 148(6): 959–970.
  3. [3] Prior, I.A., Hood, F.E., & Hartley, J.L. (2020). The Frequency of Ras Mutations in Cancer. Cancer Res. 80(14): 2969–2974.
  4. [4] Gustafsson, C., Govindarajan, S., & Minshull, J. (2004). Codon bias and heterologous protein expression. Trends Biotechnol. 22(7): 346–353.
  5. [5] Fuglsang, A. (2003). Codon optimization theory and application to expression in mammalian cells. Biotechnol. Annu. Rev. 9: 1–30.

5. 2A peptide (T2A/P2A)

- T2A(BBa_K2796007)registry.igem.org/parts/bba-k2796007
- P2A(BBa_K2796006)registry.igem.org/parts/bba-k2796006

Sequence information:

1. P2A:

GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

2. T2A:

GGAAGCGGAGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCC

<Registry Contribution>

We utilized existing iGEM Registry entries for the T2A and P2A self-cleaving peptide sequences in our mammalian expression cassette. We added notes in the Documentation section to explain how T2A and P2A can be applied in composite designs, so that future teams can refer back to the original Registry part pages and use them effectively in their own projects.

<General Information & Literature Review>

2A peptides are short self-cleaving peptides originally derived from picornaviruses, such as Thosea asigna virus (T2A) and porcine teschovirus (P2A). They function by causing ribosomal skipping during translation, allowing a single open reading frame (ORF) to generate multiple discrete proteins.

Characteristics:

  • Mechanism: The ribosome fails to form a peptide bond at the C-terminus of the 2A peptide, releasing the upstream protein while continuing to translate the downstream protein.
  • Efficiency: Co-expression using 2A peptides typically yields near-equimolar ratios of the upstream and downstream proteins, unlike IRES elements that often lead to imbalanced expression.

Design Rationale:

  • Enables expression of NRAS together with reporters (Luc2, mCherry) and selection markers (Puromycin resistance) from a single transcript.
  • Ensures balanced expression of target protein and reporter, critical for correlating NRAS expression with phenotypic readouts.
  • Keeps overall construct compact compared to IRES-based designs, which improves transfection efficiency.

<Application in Our Project>

In our iGEM project, T2A/P2A peptides were inserted between the NRAS^WT or NRAS^G12D coding sequence and downstream reporters (Luc2, mCherry) or selection markers.

  • Goal: Achieve co-expression of NRAS and fluorescent/luminescent reporters from a single transcript, ensuring phenotypic differences can be quantitatively monitored without variability in promoter strength.

Design Considerations:

  • Reporter coupling: T2A/P2A ensures that expression of Luc2 or mCherry faithfully reflects NRAS expression levels.
  • Experimental flexibility: Different 2A variants (T2A vs P2A) may be tested to optimize cleavage efficiency and minimize peptide “scar” effects.
  • Compact design: The small size of 2A sequences maintains a plasmid size (~7–8 kb) that is still amenable to efficient lipofection.
References
  1. [1] Szymczak, A.L. et al. (2004). Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide–based retroviral vector. Nat Biotechnol, 22(5): 589–594.
  2. [2] Szymczak-Workman, A.L. et al. (2012). 2A peptide-mediated multi-cistronic vectors in biomedical research. Trends Mol Med, 18(8): 384–393.
  3. [3] de Felipe, P. et al. (2006). Efficacy and limitations of 2A peptide sequences in polycistronic expression in mammalian cells. J Biol Chem, 281(20): 830–835.

6. Reporter genes (Luc2, mCherry)

- Luciferin(LUC)(BBa_K3734014)registry.igem.org/parts/bba-k3734014
- mCherry(BBa_2514ZSQ7)registry.igem.org/parts/bba-2514zsq7

1. Luc2(ns)
ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTG
2. mCherry
ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA

<Registry Contribution>

1. Luc2 (Firefly luciferase):

We utilized the existing iGEM Registry entry for the luciferin-related part in our project. To support future use, we added notes in the Documentation section describing how this sequence can be applied in design, providing context and practical guidance for implementation in experimental setups.

2. mCherry

We contributed a domesticated version of the mCherry fluorescent protein coding sequence that was modified to be RFC[10]/RFC[1000]-compatible through synonymous substitutions. The original amino acid sequence and fluorescence function were preserved, while internal restriction sites were removed. By providing this standardized version, future teams can readily insert mCherry into their constructs as a reporter, enabling convenient visualization of gene expression in diverse designs.

<General Information & Literature Review>

Reporter genes are widely used in molecular biology to enable monitoring of gene expression, protein localization, and cellular phenotypes. In our construct, we incorporated Luc2 (firefly luciferase, codon-optimized) and mCherry (monomeric red fluorescent protein) as dual reporters.

Characteristics:

  • 1. Luc2 (Firefly luciferase):
    • Derived from Photinus pyralis (firefly).
    • Catalyzes the oxidation of luciferin, producing bioluminescence measurable with a luminometer.
    • Advantages: extremely sensitive, allows non-destructive, quantitative measurements of cell viability and transcriptional activity.
    • Additional benefit: commercial vector platforms (e.g., VectorBuilder) and previous studies note that including luciferase as a co-reporter can stabilize and enhance expression of the target gene, acting as a surrogate marker of proper expression.
    • Optimized Luc2 variants are codon-optimized for mammalian expression, providing higher signal-to-noise ratio and robust expression compared to earlier luciferases.
  • 2. mCherry (fluorescent protein):
    • A red fluorescent protein engineered from Discosoma sp.
    • Excitation peak ~587 nm, emission peak ~610 nm.
    • Advantages: stable, monomeric, non-toxic; allows real-time visualization of transfected cells via fluorescence microscopy.
    • Useful for qualitative tracking of transfection efficiency and co-expression with other constructs.

Complementarity of Luc2 and mCherry:

  • Luc2: quantitative, sensitive readout (drug assays, viability, promoter activity) and indirect assurance of NRAS expression.
  • mCherry: visual, real-time confirmation of transfection and expression.
  • Together they provide both qualitative and quantitative data, and help ensure that observed phenotypes reflect NRAS activity.

Design Rationale:

  • Provide dual-level measurement: visual (fluorescence) and quantitative (luminescence).
  • Luc2 additionally acts as a proxy for NRAS expression stability, ensuring robust co-expression of NRAS^WT and NRAS^G12D.
  • Reporters are linked to NRAS via 2A peptides to ensure expression is proportional and not influenced by independent promoter variation.

<Application in Our Project>

In our iGEM project, Luc2 and mCherry reporters were coupled to the NRAS^WT and NRAS^G12D constructs using T2A/P2A peptides.

  • Goal: Enable both real-time tracking of transfected cells and quantitative assessment of NRAS-driven phenotypes, while maintaining robust co-expression of the target gene.

Design Considerations:

  • Luc2: Provides a sensitive, quantitative readout for cell viability assays, drug response experiments, and longitudinal monitoring, while simultaneously acting as a surrogate indicator of NRAS expression.
  • mCherry: Serves as a fluorescent marker for verifying transfection efficiency, imaging spheroids, and co-culture experiments.
  • Dual reporter strategy: Ensures that differences in phenotypes are attributable to NRAS mutation rather than inconsistencies in transfection efficiency.
References
  1. [1] Promega (2010). Luciferase Reporter Assays: Powerful Tools for Understanding Gene Regulation. Promega Technical Manual.
  2. [2] Thompson, J.F., & Miller, A.D. (2002). Fluorescent and luminescent reporter gene assays for biological systems. Curr Opin Biotechnol, 13(4): 289–296.
  3. [3] Shaner, N.C. et al. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol, 22(12): 1567–1572.
  4. [4] Hall, M.P. et al. (2012). Engineered luciferase reporter from deep sea shrimp utilizing a novel substrate. ACS Chem Biol, 7(11): 1848–1857.
  5. [5] VectorBuilder Technical Notes (2023). Reporter genes for enhancing and monitoring expression.

7. PolyA signal (BGH pA, SV40 late pA)

- BGH pA(BBa_J176009)registry.igem.org/parts/bba-j176009
- SSV40 late pA(BBa_K5316011)registry.igem.org/parts/bba-k5316011

Sequence information:

1. BGSH pA

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

2. SV40 late pA

CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA

<Registry Contribution>

We utilized existing iGEM Registry entries for the BGH polyadenylation signal and the SV40 late polyadenylation signal in our constructs. To support future applications, we added notes in the Documentation section describing how these polyA elements can be incorporated into mammalian expression designs, providing guidance for efficient transcription termination and mRNA stabilization.

<General Information & Literature Review>

Polyadenylation (polyA) signals are cis-acting sequences that direct the cleavage and polyadenylation of nascent transcripts in eukaryotes. This modification increases mRNA stability, facilitates nuclear export, and enhances translation efficiency.

Characteristics:

  • BGH pA (Bovine Growth Hormone polyA): Widely used in mammalian expression vectors. Provides strong transcriptional termination and efficient mRNA stabilization.
  • SV40 late pA (Simian Virus 40 polyA): Another commonly used signal derived from viral vectors. Effective in a wide range of mammalian cells, often used in dual-promoter systems.
  • Function: Both serve as robust transcriptional terminators, ensuring reliable expression of the upstream gene.
  • Safety: As non-coding regulatory sequences, polyA signals present no biosafety concerns.

Design Rationale:

  • Ensures efficient termination of transcription and prevents transcriptional read-through.
  • Improves mRNA stability, maximizing expression of NRAS constructs.
  • BGH pA was selected for our vector due to its proven high efficiency in mammalian systems.

<Application in Our Project>

In our iGEM project, the polyA signal was placed downstream of the NRAS^WT and NRAS^G12D expression cassettes to stabilize transcripts and guarantee strong expression.

  • Goal: Ensure proper transcriptional termination and mRNA stability for consistent NRAS expression.

Design Considerations:

  • Guarantees efficient polyadenylation, preventing transcriptional read-through.
  • Stabilizes NRAS transcripts, minimizing variability in expression between experimental groups.
  • Provides reliable expression levels necessary for accurate phenotypic comparisons in proliferation, viability, and migration assays.
References
  1. [1] Carswell, S., & Alwine, J.C. (1989). Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol Cell Biol, 9(10): 4248–4258.
  2. [2] Higuchi, R. et al. (1990). Stabilization of transient expression of human growth hormone cDNA by the BGH polyadenylation signal in CHO cells. Nucleic Acids Res, 18(22): 6537–6544.
  3. [3] Proudfoot, N. (2011). Ending the message: poly(A) signals then and now. Genes Dev, 25(17): 1770–1782.

8. Puromycin resistance gene (PuroR)(BBa_25XP4W10)registry.igem.org/parts/bba-25xp4w10

Sequence information:
ATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAAACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGA

<Registry Contribution>

We contributed a domesticated version of the puromycin resistance gene (PuroR) that was modified to be RFC[10]/RFC[1000]-compatible by removing internal restriction sites through synonymous substitutions. The amino acid sequence and resistance function were preserved. This standardized version allows future teams to easily incorporate puromycin resistance into their constructs, enabling efficient selection and maintenance of stably transfected mammalian cells.

<General Information & Literature Review>

Puromycin is an aminonucleoside antibiotic that inhibits protein synthesis by causing premature chain termination. Expression of the pac gene (puromycin N-acetyltransferase, abbreviated as PuroR) confers resistance by inactivating puromycin.

Characteristics:

  • Fast selection: Puromycin kills mammalian cells rapidly (1–3 days), enabling quick establishment of stable lines.
  • Marker gene: PuroR allows enrichment of transfected/transduced cells, ensuring homogeneity in experimental populations.
  • Widely validated: Commonly used in mammalian research, including cancer and stem cell models.
  • Safety: Derived from Streptomyces alboniger, non-toxic beyond its role in conferring antibiotic resistance.

Design Rationale:

  • Included in our construct to allow creation of stable NIH3T3 lines expressing NRAS^WT or NRAS^G12D.
  • Facilitates long-term experiments (drug resistance assays, 3D spheroid culture) by ensuring consistent expression across populations.
  • Used in combination with EF1α–NRAS–Luc2/mCherry modules for both functional assays and phenotypic monitoring.

<Application in Our Project>

In our iGEM project, the puromycin resistance gene was incorporated to enable stable NIH3T3 cell line generation for NRAS studies.

  • Goal: Select and maintain homogeneous cell populations expressing NRAS constructs for long-term experiments.

Design Considerations:

  • Allows rapid and efficient selection of cells harboring the complete NRAS cassette.
  • Reduces variability associated with transient transfection, improving reproducibility of results.
  • Supports extended studies, including drug-response assays and 3D spheroid modeling, by ensuring stable NRAS expression.
References
  1. [1] Vara, J.A. et al. (1986). Cloning and expression of a puromycin-N-acetyltransferase gene from Streptomyces alboniger in mammalian cells. Gene, 49(2-3): 293–298.
  2. [2] Hellebrand, E. et al. (2000). Puromycin selection of stable transfectants in mammalian cells. Biotechniques, 29(3): 590–594.
  3. [3] Ran, F.A. et al. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8(11): 2281–2308. (notes puromycin as a standard mammalian selection marker).

9. Composite Part (Final Construct)

- EF1α promoter – hNRAS^WT – mCherry – PuroR composite (BBa_25FFVYR9)registry.igem.org/parts/bba-25ffvyr9

- EF1α promoter – hNRAS^G12D – mCherry – PuroR composite (BBa_25XB94BL)registry.igem.org/parts/bba-25xb94bl

<Registry Contribution>

We created two composite parts: EF1α promoter – hNRAS^WT – mCherry – PuroR and EF1α promoter – hNRAS^G12D – mCherry – PuroR. These constructs integrate a strong mammalian promoter, NRAS coding sequences (wild-type or G12D variant), a fluorescent reporter, and an antibiotic resistance marker into modular expression cassettes. By uploading both variants, we provide future teams with valuable references for NRAS-related research and ready-to-use designs that can be adapted or recombined for diverse applications. This contribution supports the study of oncogenic mutations and enables flexible design of mammalian expression systems.

Figure 1-1
Figure 1-1 (Plasmid map of final construct)
Figure 1-2
Figure 1-2 (Plasmid map of final construct)

<General Information & Literature Review>

Composite parts integrate multiple biological elements—promoters, coding sequences, linkers, reporters, selection markers, and terminators—into a single functional unit to achieve robust expression in mammalian cells.

● In this project, the composite construct combines:

  • EF1α promoter (constitutive expression across mammalian cells).
  • Kozak sequence (efficient translation initiation).
  • hNRAS^WT or hNRAS^G12D coding sequence (oncogenic variant with codon optimization).
  • 2A peptide (T2A/P2A) (ribosomal skipping sequence enabling co-expression).
  • Luc2 or mCherry reporter (quantitative luminescence and real-time fluorescence).
  • PolyA signal (BGH pA / SV40 late pA) (mRNA stabilization and termination).
  • Puromycin resistance gene (PuroR) (rapid antibiotic selection for stable cell line establishment).
  • Plasmid backbone (pUC ori, AmpR) for bacterial cloning and amplification.

● This composite design ensures:

  • Stable and continuous expression of NRAS variants in mammalian fibroblast cells (NIH3T3).
  • Coupled expression of reporters and NRAS via 2A sequences, ensuring reporter activity reflects target gene expression.
  • Efficient transcript stability and translation provided by polyA signals and Kozak sequence.
  • Selection capability with puromycin, enabling generation of stable lines for long-term studies.

Such composite constructs are widely used in cancer modeling and functional genomics to study oncogene-driven cellular phenotypes, drug resistance, and signaling pathways.

<Application in Our Project>

In our iGEM project, this composite part represents the core experimental tool that enables the direct comparison of wild-type NRAS and the oncogenic NRAS^G12D variant in NIH3T3 cells.

  • The EF1α–NRAS–reporter cassette provides stable, constitutive expression, allowing us to analyze mutation-driven differences in growth, viability, and migration.
  • The Luc2/mCherry reporters allow dual-level monitoring: quantitative luminescence assays and qualitative fluorescence imaging.
  • The puromycin resistance marker enables stable cell line generation, critical for long-term assays and 3D spheroid modeling.

Such composite constructs are not limited to iGEM applications but can also be broadly applied for luciferase-based activity measurements, functional studies across diverse mammalian cell types, and validation of NRAS gene effects on cellular behavior. In particular, this design plays a crucial role in our 3D blood cancer platform, where it enables systematic analysis of oncogenic NRAS signaling, cell proliferation, and drug response in a physiologically relevant microenvironment.

This composite part therefore provides the functional blueprint for studying oncogenic NRAS signaling in mammalian cells, supporting applications in cancer biology, drug screening, and synthetic biology modeling.