Experiments
Arabidopsis thaliana transformation and protein purification
The plasmid with the transcriptional units GBSS-Xa-GFP and kanamycin was electroporated in an Agrobacterium tumefaciens strain harboring a disarmed Ti plasmid to transform Arabidopsis plants. The method used to transform Arabidopsis was floral dip method. This method consists in dipping Arabidopsis flower buds in an Agrobacterium cell suspension to allow uptake of the agrobacteria into female gametes. Although the overall transformation efficiency of the floral dip method may not be high, the total number of seed produced by an Arabidopsis plant ensures that sufficient transgenic events can be recovered even in a single transformation experiment.
Protein purification
In order to purify our protein, the first step was to purify starch granules from leaves. The fresh material was homogenized with a waring blender, filtered and subjected to several centrifuge and wash steps. Finally we obtained starch free of any cellular contamination.
The final objective is to obtain in an easy way the protein of interest that is fused to the starch granule (in our case GFP). To do that we digested the purified starch with factor Xa protease. This protease cut the junction between GFP and GBSS enzyme and release the protein of interest. After digestion, with a simple centrifugation step we separated the pellet (that contained starch granules) from the supernatant (that contained GFP in a high grade of purity).
Improvement of protein production by using volatile compounds
To improve the protein production, using the proposed design, quick and low-cost purification system, we implement in our project the discovery that our laboratory supervisors. From CSIC, discovered few years ago. They found that volatile compounds (VCs) emitted by the fungus Alternaria alternata improved the metabolism of any kind of plant (not only our model plant Arabidopsis but also agronomical interesting crops as pepper or maize) and, in consequence, the plants grew faster and improved starch production. Therefore, as in our project the target protein (GFP) is bound to the starch granule, if we can increase plant starch production we will also increase the protein production and as consequence the efficiency of our proposed system.
To verify to what extend this will happen, we grew our modified Arabidopsis plants, expressing GBSS-Xa-GFP (BBa_K3045002), in the presence or absence of Alternaria alternata culture. We placed the plant and the fungi in separate plates in a “box in box” system that avoid direct contact between the plant and the microorganism. One day latter, we collected and frozen all the plant leaves. Sampling moment was at the end of the photoperiod, the moment in which starch accumulated in leaves is higher. Starch and the target protein (GFP) were extracted, purified and quantified as described in the section “measurement”.
Construction of plasmid GBSS-Xa-GFP PAP1 for protein production in Mars
One of the main objectives of this project is to use plants as biofactories to produce proteins of interest in Mars. So, having in mind the extreme ambient that our plants will find in the red planet and using synthetic biology techniques we added new characteristics to our plants, in particular we included a transcriptional unit for PAP1 gene. Overexpression of this gene increase the amount of anthocyanins in plants, compounds that protect plants from high UV irradiation. The new plasmid was constructed using the cloning system GoldenBraid. The strategy that we have followed is combining two transcriptional units in one plasmid (GBSS-factor Xa protease cleavage site-GFP and PAP1) to generate the plasmid GBSS-Xa-GFP-PAP1. This new plasmid could be combined with a Part that contains a transcriptional unit for kanamycin resistance gene in a binary destiny vector and we will have a plasmid ready to transform plants with Agrobacterium tumefacines. The plants transformed with GBSS-Xa-GFP-PAP1 will produce a protein of interest easy of purify and they will be able to growth in an ambient with high levels of irradiance.
