Difference between revisions of "Part:BBa K5088618"
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===Measurement Setup=== | ===Measurement Setup=== | ||
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==Results== | ==Results== | ||
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| − | + | We successfully detected reporter gene signals for both mCherry and eGFP in most of the 3' UTR constructs during tobacco leaf infiltrations, indicating that the design of these regulatory elements is generally viable. | |
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Latest revision as of 09:39, 2 October 2024
Measurement construct - 3'UTR OCS
This composite part serves as the measurement construct for the basic part BBa_K5088120 (OCS).
Contents
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 2704
Illegal PstI site found at 3131 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 3131
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 230
Illegal BglII site found at 2707 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 2704
Illegal PstI site found at 3131 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 2704
Illegal PstI site found at 3131
Illegal NgoMIV site found at 1882
Illegal NgoMIV site found at 2179
Illegal AgeI site found at 1755 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2393
Testing & Measurement
Dual Fluorescence Reporter Assay
Experiments in plant biology are often susceptible to high variability, with factors like transformation efficiency, cell viability, and environmental conditions contributing to noisy results.
To mitigate these issues, we employed a dual fluorescence reporter assay. In this setup, both the target and reference proteins are expressed from the same plasmid, allowing for precise and reliable characterization.
Using a second, constant fluorescence reporter as an internal reference allows us to normalize the readout, ensuring that observed fluorescence accurately reflect the activity of the regulatory elements under study, independent of external factors.
Figure 13: SBOL scheme of our Tarakate - consensus measurement construct [BBa_K5088677].
Choice of Reporter Genes
For our initial round of testing, we evaluated a construct design using the plant GFP from the iGEM distribution kit as a reporter system.
In our initial experiments, we were unable to detect any fluorescence in dandelion leaf infiltrations or protoplast transformations. To troubleshoot, we tested the construct in tobacco leaf infiltrations. This process required several rounds of protocol optimization before we finally achieved positive GFP expression in tobacco leaves. After further adjustments, we also obtained very low GFP signals in dandelion protoplasts.
Despite these optimizations, the signal strength remained weak, registering only about three times above background levels. This indicated that the system was still not sensitive enough for effective promoter characterization. The avGFP from the iGEM distribution kit, an older and less bright variant, likely contributed to the low signal observed.
To address this, we switched to a more advanced GFP variant, eGFP, known for its higher brightness. Moreover we did extensive literature research and found out that Agrobacterium is able to use some plant promoters, leading to fluorescence that would be indistinguishable from the plant tissue"s expression.
Therefore we decided to introduce the potato ST-LS1 intron into the eGFP coding sequence to prevent Agrobacterium from expressing GFP, as this has been described as a suitable strategy for that problem.
A further challenge we encountered was the variability in transformation efficiency during Agrobacterium-mediated leaf infiltration and protoplast transformation. This variability made it difficult to perform quantitative measurements of the individual regulatory parts across biological replicates. To overcome this issue, we adopted the previously reported ratiometric approach by incorporating a second reporter gene in each construct. This reference reporter is located on the same plasmid as the GFP to minimize noise and provide a reliable normalization standard for more accurate quantitative analysis. To read more about the optimization of our reporter construct visit our engineering page
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 14: 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.
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- Plate reader
- 96-well or 384-well plate black, clear flat bottom
- Forceps
- 0.7 mm cork borer
Protocol for Leaf Disk Measurement
- Fill every well with 10µl ddH2O
- Punch out the leaf disks using a cork borer. Then, place them with a forceps in the 96-well plate abaxial side down
- Make sure to close the lid in between to prevent dehydration
- We performed all ratiometric leaf disk assays in a plate reader (BMG labtech, CLARIOstar Plus)
- Settings for eGFP (Ex: 472nm ± 6nm, Em: 515 ± 6nm)
- Settings for mCherry (Ex: 570nm ± 6nm, Em: 610nm ± 6nm)
Figure 16: 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.
Results
We successfully detected reporter gene signals for both mCherry and eGFP in most of the 3' UTR constructs during tobacco leaf infiltrations, indicating that the design of these regulatory elements is generally viable.
Figure: Leaf infiltration assay using Nicotiana benthamiana. Shown are (A) eGFP expression, (B) mCherry expression and (C) ratiometric between both reporters.
The Dandelion Toolbox
Our project aimed to advance the genetic engineering of dandelions by developing a robust set of constitutive regulatory parts. Using a transcriptomic approach, we identified 40 endogenous elements. To ensure precise and reliable testing, we constructed a ratiometric measurement system, enabling effective and quantitative characterization of these parts.
We employed three distinct plant transformation methods to test and validate the functionality of the regulatory elements. Through rigorous testing, we successfully characterized 23 out of the initial 40 elements, resulting in a comprehensive collection of standardized dandelion parts. This well-characterized suite of parts is designed to streamline future complex genetic engineering projects.
By providing these standardized tools, our project significantly lowers the barriers for researchers and iGEM teams, making Taraxacum kok-saghyz a more accessible and versatile chassis for plant synthetic biology. Ultimately, our work contributes to enhancing dandelion as a model organism and supporting sustainable natural rubber production.
Overview
| 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 |
Dandelion Handbook
By creating a suite of genetic tools and transformation methods, and sharing them through our Dandelion Handbook, we believe that dandelions can serve as an excellent chassis for numerous applications. We aim to inspire future iGEM teams to harness the unique properties of dandelions for a variety of promising projects.
Dandelions have demonstrated their versatility, being used as a coffee alternative and in various food applications such as salads, wine, and honey. Additionally, their ability to naturally hyperaccumulate environmental pollutants, including heavy metals, highlights their potential for bioremediation applications.
By equipping future iGEM teams with these resources, we aspire to unlock the full potential of dandelions, paving the way for sustainable and diverse synthetic biology applications.
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