Difference between revisions of "Part:BBa K5088150"

Revision as of 05:50, 2 October 2024

Plant GFP with IV2 intron

View the results of this basic part.
Check out the corresponding measurement construct by clicking here.

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.

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.”

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.

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).

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

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)

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)

Results

Leaf Infiltration

Taraxacum

N. benthamiana

Protoplast

Cut-Dip-Budding