Part:BBa_K5466001

IGG6/NAL10

First called IGG6, then renamed NAL10: A 9-bp sequence that enables polycistronic gene expression in fungi, yeasts and other filamentous fungi, plants and animals. Identified and characterized by Yue et al. (2023), it has been proven functional for bicistronic expression in the yeasts Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, as well as the filamentous fungus Aspergillus nidulans. IGG6 has shown polycistronic expression up to 4 CDSs in Saccharomyces cerevisiae.

In Ma et al., (2024) IGG6 sequence was demonstrated to be functional in organisms further from fungi. By generating a NAL10-mediated bicistron expressing mCherry and GFP with distinct localization signals, fluorescent intensities were shown to localize to different cellular subcompartments both in maize protoplasts and human 293 T cells.

Figure 1: Overview of IGG6-mediated polycistronic expression: the IGG6 sequence promotes translation re-initiation by the ribosome, permitting the separate translation of consecutive CDSs in an mRNA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Origin and properties of IGG6

Synthetic biology increasingly demands the coexpression of a large number of genes in order to achieve desired functions. To reduce the length and complexity of genetic constructs, systems for polycistronic expression are required, permitting the expression of several genes from a single promoter and avoiding a multiple promoter strategy.

Well established systems are IRES (internal ribosome entry sites) and 2A peptides. Although functional, these sequences have their downsides. IRES have a limited efficiency and they are generally long in sequence. 2A peptides have a greater efficiency, although their mechanism implies the modification of the product proteins through the append of amino acids, which may lead to altered structure and impaired function, as well as interference with signal peptides functio.

IGG6 is an optimization of IGG1, an intergenic sequence that generates functional operons in Glarea lozoyensis. It has shown properties that make it a grate candidate for polycistronic expression in eukaryote:

IGG6 is functional in various species

Yue et al. (2023) achieved to generate GFP bicistrons in various fungal species: TDH3::IGG6-GFP in Pichia pastoris, TEF1::IGG6-GFP in Yarrowia lipolytica, NpgA::IGG6-GFP in Aspergillus nidulans. GFP expression was verified in all cases through fluorescence microscopy.

Figure 2: Fluorescence microscopy of GFP-bicistron-expressing strains of various fungi. Figure from Yue et al. (2023).

IGG6 supports polycistronic expression in Saccharomyces cerevisiae

IGG6-mediated expression of the zeocin-resistance gene (KanR) was achieved, providing resistance when positioned up to the fourth CDS. At the same time, the expression of the rest of the CDSs was proven by β-carotene production through the introduction of the carotenoid biosynthetic genes crtYB, crtE, crtI. IGG6-efficiency is maximal for the second CDS and expression decays progressively from the third position.

Figure 3: Cell growth in the presence of zeocin of strains expressing KanR gene at different positions of a polycistronic expression cassette. Figure from Yue et al.(2023).

IGG6 produces separate unfused proteins

When IGG6 was used to genrate a bicistron expressing mCherry and GFP with two different subcellular localization signal, targeting the peroxisome (MDH3) and nucleus (SV40 NLS) respectively, each fluorescence signal was only observed in the expected subcompartment. Instead, when a fusion protein was synthesized, both signal colocalized.

Figure 4: Fluorescence microscopy of IGG6-mediated mCherry-MDH3 and GFP-SV40 NLS-coexpressing strain. Figure from Yue et al. (2023).

IGG6 mediates translation re-initiation

Regarding the mechanism of IGG6, Yue et al. (2023) designed a series of bicistronic constructs harboring TDH3-FLAG followed by the GFP gene, with or without a translation blocking sequence (TBS) in different parts of the construct. TBS prevented GFP expression when positioned anywhere in the construct upstream of the GFP gene, demonstrating that translation of the message downstream of IGG6 is dependent on the translation of the upstream coding sequence. Based on this evidence, a mechanism of translation re-initiation was proposed for IGG6, where the ribosome might not dissociate and would begin scanning for the following start codon.

Figure 5: Translation re-initiation at the distal gene GFP, mediated by IGG6. Translation of Tdh3p and GFP from different gene expression units was monitored by Western blotting and fluorescence microscopy, respectively. The scale bars for the images are 20 μm. Red triangles represent a translation blocking sequence (TBS). Figure from Yue et al. (2023).

IGG6/NAL10 is functional in plants and animal

In maize protoplast, NAL10 was used to produce mCherry with a Golgi localization signal and GFP with a nuclear localization signal.

Figure 6: Confocal microscopy of maize protoplast expressing GLS-mCherry-NAL10-NLS-GFP cassette. Figure from Ma et al. (2024).

In human 293 T cells, a bicistron was implemented producing GFP and mCherry with SV40 NLS. Green fluorescent was visible in the whole cytoplasm while red fluorescence localized to the nucleus.

Figure 7: Human 293 T cells expressing mCherry-NAL10-NLS-GFP cassette. Figure from Ma et al. (2024).

IGG6 is competitive against previosly utilized IRES and 2A peptides

IGG6 performance was compared to six IRES with high ribosome-recruiting activities and two highly efficient 2A peptides for the production of GFP-bicistrons. Based on the fluorescence intensity of the resulting strains, IGG6 was shown to outperform IRES with a 12 to 130-fold increase in signal, while falling closely behind 2A peptides with 37-47% of the fluorescence intensity.

Figure 8: GFP-signal from IRES, 2A peptides and IGG6-mediated bicistrons in Saccharomices cerevisiae. Six IRES (IRES8, IRES10, IRES32, IRES40, IRES41, IRES47) and two 2A peptides (ERBV2A, P2A) were chosen for the assay. Figure from Yue et al. (2023).

Therefore, IGG6/NAL10-based polycistronic constitutes a great alternative to the existing methods due to its high efficiency and the unmodified sequence of the proteins produced.

Aplications

The properties of this novel polycistronic expression system confers the potential to revolutionize several aspects of synthetic biology and new technologies based on this system are being developed.

In the context of metabolic engineering, the development of polycistronic genes in fungi, plants and animals can simplify the implementation of intricate and complex metabolic pathways. The successful introduction of a new pathway requires the fine-tuning of protein expression and ensurance of stable levels of production. Yue et al. (2023) developed a system called HACKing (Highly efficient and Accessible system by CracKing genes into the genome). Utilizing a library of 65 validated driver genes, they employed GTR-CRISPR21 for the rapid, multiplexed integration of multiple genes of interest (GOIs) into bicistronic transcription units, eliminating the need for selection markers and facilitating the simultaneous expression of GOI-encoded enzymes at stable, pre-calibrated levels. The HACKing system was validated through the rapid and efficient creation of a S. cerevisiae strain that produces high amounts of triterpene squalene.

Figure 9: HACKing system: A host gene with an appropriate, pre-validated translation level under the desired cultivation conditions is selected for each GOI to serve as a driver. Figure from Ma et al. (2024).

IGG6/NAL10 also facilitates gene stacking, which combines multiple genes to enhance traits such as disease resistance, crop productivity, or other desirable horticultural characteristics in plants. In humans, it enables the simultaneous delivery of several therapeutic agents necessary for treating diseases.

Examples using this part

First, we present an example involving three fluorescent proteins (BBa_K5466033) designed to serve as a preliminary test of functionality, providing a foundation for future teams to explore and characterize IGG6 in greater detail. Thanks to the iGEM Fluorescence Measurement Calibrants Kit, it is possible to convert OD600 and fluorescence intensity units into absolute units. This would allow for the comparison of the effect of position in a polycistronic gene, thanks to IGG6, on translation.

Second, we present an example of a multi-response mechanism mediated by IGG6 (BBa_K5466020), which includes the following components:

• Expression of Nb28-S102D Aga2P: This is a VHH with a high affinity for AFB1, anchored to the cell wall, enabling the capture of AFB1 present in the environment.

• Positive Feedback Loop: This mechanism amplifies the response, ensuring that upon the initial detection of aflatoxin, the maximum response is triggered. This leads to saturation of the yeast cell wall with VHH, thereby sustaining the response over time. The positive feedback occurs because the transcription factor that activates the expression of this device is synthesized.

• Reporter RFP: This reporter allows us to visualize the presence of aflatoxin in the environment, confirming that the response is indeed occurring.

Lastly, we demonstrate that IGG6 facilitates a reduction in the size of the genetic construct required for expression of our signaling platform for detecting AFB1 with (BBa_K5466023). Typically, 5558 bp would be needed to express our signaling platform for detecting AFB1, which requires the use of two promoters, two CDS (antiAFB1-scFv1 NubG and antiAFB1-scFv2 Cub) along with two terminators. However, with IGG6, we can reduce this by approximately 1000 bp, needing only one promoter, antiAFB1-scFv1 NubG, IGG6, antiAFB1-scFv2 Cub, and one terminator, resulting in a total of 4647 bp.

Considerations when using this part

A common practice in synthetic biology is to add two stop codons to the CDSs; if you wish to use this part, it is recommended to use only one stop codon. Starting from the second stop codon, experiments with fluorescent proteins show that the signal gradually weakens until it disappears at the third stop codon.

References

Ma, X., Yue, Q., Miao, L., Li, S., Tian, J., Si, W., Zhang, L., Yang, W., Zhou, X., Zhang, J., Chen, R., Xu, Y., & Liu, X. (2024). A novel nucleic acid linker for multi‐gene expression enhances plant and animal synthetic biology. The Plant Journal, 118(6), 1864–1871. https://doi.org/10.1111/tpj.16714

Yue, Q., Meng, J., Qiu, Y., Yin, M., Zhang, L., Zhou, W., An, Z., Liu, Z., Yuan, Q., Sun, W., Li, C., Zhao, H., Molnár, I., Xu, Y., & Shi, S. (2023). A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-40027-0

Wang, X., & Marchisio, M. A. (2021). Synthetic polycistronic sequences in eukaryotes. Synthetic and systems biotechnology, 6(4), 254–261. https://doi.org/10.1016/j.synbio.2021.09.003