Goals
The project's main aim is to create specialized plants, known as biosensors, that can identify specific substances. For this initial test run, we've chosen to focus on detecting carbon dioxide (CO2).
Another goal is to develop new designs that enhance the effectiveness of the ones you've been using. To achieve this, we're incorporating different marker genes, which are like labels, with advantages that are better than what you get from GFP (a common marker), and a system that causes the signal from the marker protein to disappear when the stimulus is no longer present. This makes the whole process smarter and more efficient.
This year we are working with the 4 selected promoters, and we are following the project by researching genes for the codificant region of the transcriptional unit.
- For this task we have developed three lines of investigation to make the project viable CO2 inducible promoters with the Gateway Cloning Method.
- Adding visible reporter genes (EYGFPuV and RUBY) to our construction with the Gateway Cloning Method.
- Adding protein degradation sequences (PEST sequence) to our construction with the Golden Braid Technology.
The biological engineering in each part is not always the same, due to the complexity of the task we have used two, the Gateway Cloning Method and the Golden Braid Golden Method.
Genetically modify Arabidopsis thaliana using CO2-responsive promoters from last year, employing the Gateway method.
Design
Gateway method
The Gateway Cloning Method is a powerful and flexible molecular biology method advanced through Invitrogen for the efficient and unique cloning of DNA fragments into plasmid vectors. It simplifies the manner of moving genes or DNA fragments among unique plasmid vectors without the need for traditional restriction enzyme-based totally cloning. The Gateway Cloning Method is broadly utilized in molecular biology and Synbio research and has turned out to be a trendy technique in many laboratories.
Here's an overview of how the Gateway Cloning Method works:
Create Entry Clones: In this step, you begin by using amplifying your DNA fragment of hobby (e.g., a gene) using PCR. You then introduce DNA recombination websites referred to as "attB websites" to the PCR product. These attB websites are recognized with the aid of special recombination enzymes.
BP Recombination: After developing Entry Clones, you perform a BP (Gateway BP) recombination response. This response includes blending the Entry Clone (containing your DNA fragment with attB sites) with a plasmid vector containing attP sites. The BP Clonase enzyme recognizes those websites and allows recombination. This affects the transfer of your DNA fragment into the destination vector, which is referred to as a "Donor Vector." The resulting Donor Vector now incorporates your DNA fragment flanked with the aid of attL websites. It is called the BP reaction because the B-s are changed with the P-s
LR Recombination: In the next step, you perform an LR (Gateway LR) recombination reaction. You mix the Donor Vector (containing your DNA fragment) with a destination vector that includes attR websites. The LR Clonase enzyme acknowledges these sites and catalyses recombination. This step transfers your DNA fragment from the Donor Vector to the Destination Vector, resulting for your preferred expression construct.
Transform Host Cells: After the LR recombination, the resulting Destination Vector, which now contains your DNA fragment beneath the manipulate of a promoter of your choice, can be transformed into a suitable host organism, in our case we introduce it inside Agrobacterium tumefaciens for plant transformation.
The Gateway Cloning Method offers several benefits, such as excessive cloning performance, no want for restrict enzymes or ligase, and the capacity to hastily assemble complex expression constructs with a couple of components. It is especially useful for excessive-throughput and modular cloning initiatives, wherein you need to transport DNA fragments among exceptional vectors effectively.
In our case, we have used this method twice, more specifically in the two first lines of research.
In the first line, we introduced our promoters inside the plasmid with the GFP and the terminator. Afterward, the construction was introduced in Agrobacterium tumefaciens with no capability of forming tumours in plants. Next, we submerge the solution of the modified bacteria in the ovules of Arabidopsis thaliana, so it passes the modification to the next filial generation.
In the case of the next line of investigation, we did it in two rounds. First, we introduced the promoter and the reporter in two different pDONRS, going from a linear sequence to a plasmid one. Then, in the second round we fused both plasmids using the LR CLonase.
So, our final constructions are plasmids with the promoters, the visible reporters (RUBY and eYGFPuV) and the terminator, TNos.
Goldenbraid method
The GoldenBraid (GB) technique is a modular and versatile DNA meeting technique utilized in synthetic biology for the development of genetic circuits and multigene constructs. Developed at the Centre for Research in Agricultural Genomics (CRAG) in Spain, the GoldenBraid technique presents researchers with an effective tool for assembling and manipulating DNA parts, inclusive of genes, promoters, and different genetic factors, to create complex organic structures.
Its technique is based in:
Type IIs Restriction Enzymes: GoldenBraid is based on Type IIs restrict enzymes, which cut DNA sequences outside in their popularity site. This characteristic is essential for unique and scarless DNA meeting. Common Type IIs enzymes used in GoldenBraid consist of BsaI and BsmBI.
Standardized DNA Parts: DNA components used in GoldenBraid are usually flanked via Type IIs enzyme popularity sites and are standardized in terms of length and structure. These elements can encompass promoters, coding sequences, terminators, and other genetic elements.
Entry Vectors: GoldenBraid uses Entry Vectors, which are circular plasmids that hold person DNA elements. These Entry Vectors have standardized cloning websites and are designed to accept DNA fragments generated through Type IIs restrict enzyme digestion.
In our project we used the GB tech in the third line of investigation (PEST DNA sequences) to research protein degradation. This method is based on developing small plasmids known as PUPD2 with the different subunits of our transcriptional unit and fusing them using the enzymes BSal and DNA Ligase.
Our final plasmid will have the name pDGB1 a1 p35S:YGFP-PEST:Tnos, in this case we have used only eYGFPuV. Giving as a result the following plasmid.
Practical demostration
Our structures have been effectively assembled using the described methods.
Subsequently, these constructs were introduced into Arabidopsis thaliana, the plant designated for modification. This plant has shown a correct response to the transformation, resulting in varying levels of GFP expression depending on the promoter used. Additionally, the genetically modified organisms (GMOs) containing the RUBY marker have also proven successful, causing the plant to exhibit a red coloration in response to the promoter. In summary, the entire experiment was a triumph, as we achieved the anticipated results successfully.