Part:BBa_K5088150

Plant GFP with IV2 intron

Assembly Compatibility:

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  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
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  COMPATIBLE WITH RFC[1000]

Background

To improve the sensitivity and reliability of our part characterization, we transitioned from using the original Aequorea victoria GFP (avGFP) - which was provided in the iGEM distribution kit - to an enhanced variant, eGFP. The avGFP, isolated in 1962, exhibited lower brightness, which likely contributed to the suboptimal signal observed in our experiments. In contrast, eGFP is a modern, brighter fluorescent protein that significantly enhances assay performance by providing a stronger and more detectable fluorescence signal.

Additionally, to mitigate the risk of false-positive fluorescence arising from unintended transcription in Agrobacterium, we incorporated the potato ST-LS1 intron (IV-2 intron) into the eGFP coding sequence. This intron effectively prevents Agrobacterium from expressing the eGFP, as plant promoters in Agrobacterium can inadvertently initiate transcription, leading to misleading fluorescence signals. The insertion of the IV-2 intron ensures that eGFP expression is restricted to plant tissues, thereby enhancing the specificity and accuracy of our fluorescence measurements.

Parts we have tested using this reporter:

Part Identifier	Part Type	Nickname	Part Description
BBa_K5088001	Promoter + 5'UTR	P_RPL28	Large subunit ribosomal protein L28e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088006	Promoter + 5'UTR	P_FKBP4_5	FK506-binding protein 4/5 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088007	Promoter + 5'UTR	P_CLTC	Clathrin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088008	Promoter + 5'UTR	P_RPL31	Large subunit ribosomal protein L31e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088012	Promoter + 5'UTR	P_Tubulin	Tubulin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088013	Promoter + 5'UTR	P_EIF5A	Translation initiation factor 5A - Promoter+5'UTR from T. kok-saghyz
BBa_K5088102	3'UTR	T_PTI1	Protein tyrosine kinase - 3'UTR from T. kok-saghyz
BBa_K5088103	3'UTR	T_RPL28	Large subunit ribosomal protein L28e - 3'UTR from T. kok-saghyz
BBa_K5088104	3'UTR	T_EPS15	Epidermal growth factor receptor substrate 15 - 3'UTR from T. kok-saghyz
BBa_K5088105	3'UTR	T_GSK3B	Glycogen synthase kinase 3 - 3'UTR from T. kok-saghyz
BBa_K5088106	3'UTR	T_MGRN1	E3 ubiquitin-protein ligase - 3'UTR from T. kok-saghyz
BBa_K5088107	3'UTR	T_RPL35A	Large subunit ribosomal protein L35Ae - 3'UTR from T. kok-saghyz
BBa_K5088108	3'UTR	T_betB	Betaine-aldehyde dehydrogenase - 3'UTR from T. kok-saghyz
BBa_K5088109	3'UTR	T_pgm	Phosphoglucomutase - 3'UTR from T. kok-saghyz
BBa_K5088110	3'UTR	T_ATP-synt	ATPase subunit gamma - 3'UTR from T. kok-saghyz
BBa_K5088111	3'UTR	T_EIF3B	Translation initiation factor 3 subunit B - 3'UTR from T. kok-saghyz
BBa_K5088112	3'UTR	T_RPL31	Large subunit ribosomal protein L31e - 3'UTR from T. kok-saghyz
BBa_K5088113	3'UTR	T_TM9SF2_4	Transmembrane 9 superfamily member 2/4 - 3'UTR from T. kok-saghyz
BBa_K5088114	3'UTR	T_CUL1	Cullin - 3'UTR from T. kok-saghyz
BBa_K5088115	3'UTR	T_PSMB6	20S proteasome subunit beta 1 - 3'UTR from T. kok-saghyz
BBa_K5088116	3'UTR	T_RPSA	Small subunit ribosomal protein SAe - 3'UTR from T. kok-saghyz
BBa_K5088117	3'UTR	T_VPS4	Vacuolar protein-sorting-associated protein 4 - 3'UTR from T. kok-saghyz
BBa_K5088118	3'UTR	T_EIF2S3	Translation initiation factor 2 subunit 3 - 3'UTR from T. kok-saghyz

Table 1: List of functioning T. kok-saghyz endogenous regulatory elements we've characterized using this reporter.

Transient Transformation

The characterization of genetic parts is crucial for understanding their functionality and behavior within a host organism. However, this process presents significant challenges, particularly when working with plants, due to the time-intensive nature of stable transformation. Stable transformation involves the integration of foreign DNA into the host genome, ensuring that the genetic modifications are inherited by subsequent generations. While this approach is crucial for characterizing the parts in their intended context, it also comes with certain drawbacks.

The process of developing stable transgenic lines is time-consuming and resource-intensive due to the need for multiple homozygous lines to ensure consistent and detectable transgene expression, which is also influenced by local genetic context. This can be especially challenging for iGEM projects. To address these challenges, transient transformation offers a faster and more efficient alternative for the rapid characterization of genetic parts.

Transient transformation enables temporary expression of a reporter construct without the need for stable integration into the host genome, significantly reducing the time required to obtain results and allowing for faster iteration and optimization of genetic constructs. Techniques such as leaf infiltration and protoplast assays are already standard in plant biology when working with model organisms like Nicotiana benthamiana. We opted to use these methods in Taraxacum and optimize these methods in order to test genetic constructs in an easier fashion.

Leaf Infiltration

Leaf infiltration is a technique used to introduce foreign DNA, proteins, or molecules into plant tissues, enabling transient gene expression without stable transformation and commonly used in Nicotiana benthamiana. It involves infiltrating a solution to the leaf surface, allowing for rapid, non-permanent expression of target genes. This method offers several benefits, including fast results, cost-effectiveness by bypassing the need for transgenic plants, and versatility in testing gene constructs or protein interactions.

We utilized the method outlined in “Genetic transformation technologies for the common dandelion, Taraxacum officinale.”

Figure 1: Workflow illustration for leaf infiltration in Taraxacum species.

Experiments were conducted using Agrobacterium rhizogenes K599 and the RNA silencing suppressor P19 in Agrobacterium tumefaciens GV3101. Both strains were cultured to an optical density (OD600) of 0.6–0.8, harvested by centrifugation, and washed twice with infiltration buffer to remove proteins from Agrobacterium that stress the plants and hinder the transformation process. The infiltration solution was adjusted to an OD600 of 0.1 in infiltration buffer, supplemented with 10 µM acetosyringone, and incubated in the dark for 3 hours.

Three- to four-week-old Taraxacum plants were infiltrated by injecting the solution into the abaxial surface of the leaves using a 1 mL syringe without a needle. Four days post-infiltration, the plants were screened for gene expression to assess transformation efficiency (Figure 2). Read more detailed protocol here: Leaf infiltration protocol.

Additionally, we modified the protocol by heavily watering the plants and covering it with a hood for 3 hours prior to infiltration to facilitate the opening of leaf stomata, thus enhancing the ease of infiltration.

Protoplast Transformation

Protoplast transformation introduces foreign DNA into plant cells without cell walls, enabling rapid gene expression and precise genetic modification. It offers high efficiency and allows researchers to study cellular processes in a controlled environment, making it a key tool in functional genomics. Furthermore, it is possible to regenerate whole transgenic plants from transformed protoplasts by culturing them in a suitable regeneration medium supplemented with specific hormones that promote cell division, callus formation, and shoot regeneration.

As a starting point, we received guidance from Prof Dr Dirk Prüfer, who provided a method for efficient protoplast isolation. For protoplast transfection, we employed techniques described in the publications “Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis” and “Genetic transformation technologies for the common dandelion, Taraxacum officinale.” We further refined these methods through multiple cycles of optimization to adapt them for our system.

Figure 2: Illustration of Taraxacum protoplast transformation >

Our experiment utilized four-week-old Taraxacum plants, where the third to fifth leaves were gently scratched on the abaxial side to remove the outer cell layer. The leaves were then submerged in 10 mL of enzyme solution (1% cellulase R-10 and 0,5% macerozyme R-10) and incubated in the dark at 30°C for 4 hours, with gentle swirling indicating protoplast release. The mixture was filtered through a 100 µm cell strainer and centrifuged, resulting in floating protoplasts, enzyme solution, and cell debris. The debris and enzyme solution were removed, and the protoplasts were washed and counted using a hemacytometer.

For transfection, polyethylene glycol (PEG) was used with 20 µg of plasmid DNA and 20,000 protoplasts. After a 15-minute dark incubation, the mixture was transferred to a protoplast-optimized solution. Gene expression in the transformed protoplasts was observed between 15 and 24 hours post-transfection.

Read more detailed protocol here: Protoplast transformation protocol.

We determined that the number of protoplasts increased with extended incubation times of Taraxacum leaves in the enzyme solution. However, a 4-hour incubation was optimal, as longer durations led to reduced protoplast viability. To minimize damage from protoplast proteases, we maintained the protoplasts on ice during all washing steps. Additionally, we observed higher transformation efficiency when the protoplasts were incubated in the dark during the transfection process, likely due to reduced stress and activation of defense mechanisms in the protoplasts.

Measurement Setup

Leaf Disk Measurement

A challenge of leaf infiltration and protoplast assays is the variability in transformation efficiency, complicating quantitative measurements across biological replicates. To solve this, we adopted a ratiometric approach, incorporating mCherry as a secondary reporter driven by the Arabidopsis ubiquitin promoter, allowing for normalization and more accurate quantification (2).

Materials

1. Plate reader
2. 96-well or 384-well plate black, clear flat bottom
3. Forceps
4. 0.7 mm cork borer

Protocol for Leaf Disk Measurement

1. Fill every well with 10µl ddH2O
2. Punch out the leaf disks using a cork borer. Then, place them with a forceps in the 96-well plate abaxial side down
3. Make sure to close the lid in between to prevent dehydration
4. We performed all ratiometric leaf disk assays in a plate reader (BMG labtech, CLARIOstar Plus)
5. Settings for eGFP (Ex: 472nm ± 6nm, Em: 515 ± 6nm)
6. Settings for mCherry (Ex: 570nm ± 6nm, Em: 610nm ± 6nm)

Figure 3: Schematic representation of the ratiometric measurement system used for the characterization of regulatory parts in T. kok-saghyz. The process begins with (1) the preparation of the infiltration solution, followed by (2) leaf infiltration using a syringe. After (3) a 4-day incubation period, leaf disks are (4) punched out and placed in a 96-well plate. (5) The samples are measured using a plate reader, and (6) ratiometric analysis is conducted, comparing eGFP and mCherry signals for quantitative characterization of the regulatory parts.

Protoplast Measurement

Protocol for Protoplast Measurement

1. All ratiometric protoplast measurements were performed using a plate reader (TECAN INFINITE M PLEX)
2. Measure autofluorescence (Ex 472 nm, Em 685 nm)
3. Measure mCherry (Ex 570 nm, Em 610 nm)
4. Measure eGFP (Ex 480 nm, Em 515 nm)

Figure 4: Schematic illustration of protoplast assay platereader measurement.

Results

Leaf Infiltration

Taraxacum

Figure 5: Fluorescence bino images (BF= Brightfield, AF= Autofluorescence Ex: Em:, mCherry Ex: Em: , eGFP Ex: Em: ) A) WT TKS, showing no mCherry and eGFP expression. B)Transgenic TKS leaves, infiltrated with Tarakate - Consensus measurement construct [BBa_K5088677]. C) Transgenic TO leaves, infiltrated with Tarakate - Consensus measurement construct [BBa_K5088677].

N. benthamiana

Figure 6: Comparison between Tarakate - Test construct GFP [BBa_K5088675] and Tarakate - Consensus measurement construct [BBa_K5088677]. The control represents non infiltrated leaf disk of N. benthamiana, K599 show leaf disks infiltrated with A. rhizogenes K599 and A. tumefaciens GV3101 P19 as additional negative controls. A) avGFP expression [Ex: 408nm ± 6nm; Em: 515nm ± 6nm] B) mCherry expression [Ex: 570nm ± 6nm; Em: 610nm ± 6nm] C) eGFP expression [Ex: 472nm ± 6nm; Em: 515nm ± 6nm].

Figure 7: Measurement of Promoter + 5'UTR parts in leaf disk assay of N. benthamiana. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Figure 8: Measurement of 3'UTR parts in leaf disk assay of N. benthamiana. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Protoplast

GFP signal for most 3' UTR constructs in both T. kok-saghyz and T. officinale, can successfully be detected via plate reader measurements of the transiently transformed protoplasts demonstrating reliable expression.

Figure 9: Fluorescense microscopy images (Nikon Ti, camera: Andor Zyla VSC-01427, objective: Plan APO VC 20x CIC N2, mCherry: Ex 575 nm, Em 647 nm, eGFP: Ex 470 nm, Em 525 nm). A) WT TKS protoplasts, showing no mCherry and eGFP expression. B) Transgenic TKS protoplasts, transfected with BBa_K5088614, showing mCherry and eGFP expression 17h post transfection.

Figure 10: Measurement of 3'UTR parts in protoplasts of T. kok-saghyz. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Figure 11: Measurement of 3'UTR parts in protoplasts of T. officinale. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.

Cut-Dip-Budding

Cut-dip-budding (CDB) is an innovative plant transformation technique that involves immersing a cut segment of a plant in a transformation solution enriched with Agrobacterium. This process effectively facilitates the introduction of desired genetic material into the plant cells, enabling efficient genetic modification. The benefits of the CDB method include its ease of use and reduced labor requirements compared to traditional transformation techniques (Cao et al., 2023a). The CDB has been optimized for Taraxacum with a stable transformation method called "Extremely simplified cut-dip-budding," recently reported in „Extremely simplified cut-dip-budding method for genetic transformation and gene editing in Taraxacum kok-saghyz“ to regenerate stably transformed dandelion shoots within 14 days without requiring plant tissue culture or specialized equipment (Cao et al., 2023b).

This is an even faster stable transformation protocol than for model organism like Arabidopsis thalianaand easy to implement for iGEM teams, since it is not sterile and straight-forward.

we selected two different promoter + 5' UTR constructs as a proof of concept to assess their ability to drive gene expression in stable transformations and across various tissues beyond leaves. We applied our rapid cut-dip protocol and successfully generated stably transformed dandelion roots. Fluorescence microscopy revealed clear signals of mCherry and eGFP in the transformed roots, confirming that the tubulin promoter + 5' UTR is functional in stable transformations.

Figure 12: Microscopy image of Cut dip budding transformed Taraxacum roots. Each subfigure is subdivided into four panels, where BF stands for brightfield and AF for autofluorescence. Shown below is the according mCherry and eGFP signal of the same root. A) Negative control B) Tarakate - Consensus measurement construct [BBa_K5088677] in T. kok-saghyz C) Tarakate - Consensus measurement construct [BBa_K5088677] in T. officinale

Figure 13:Microscopy image of Cut dip budding transformed Taraxacum roots. Each subfigure is subdivided into four panels, where BF stands for brightfield and AF for autofluorescence. Shown below is the according mCherry and eGFP signal of the same root. A) Negative control of T. kok-saghyz B) Measurement construct - Tubulin promoter+5'UTR from T. kok-saghyz [BBa_K5088512] tested in T. kok-saghyz.