Selection of CO2 plant biosensors
Arabidopsis thaliana with CO2 inducible promoters
After carrying out the permanent transformation of Arabidopsis thaliana plants, with different CO2 inducible promoters and the GFP as reporter gene, we obtained several independent lines of each construct.
We exposed these plants to high and ambient CO2 (control plants) for 6 and 24 hours and measured the expression of GFP in both CO2 conditions.
In order to select the best line of each construction, to be used as CO2 biosensors, we have made two sequential experiments with their corresponding GFP quantification measurements:
- In the first experiment, we test several the lines for each of the CO2 inducible promoters and measured the activation of the promoter in high CO2 by quantifying GFP content.
- Based on the results obtained in the first experiment, we selected the best line for each construction that respond to high CO2 concentration and measured the activation of the promoter in high CO2 by quantifying GFP content.
The CO2 inducible promoters selected to our proof of concept were ADH1, ATPS, CYS4 y G3PDH.
To quantify the GFP properly and to obtain comparative data, the first thing we did was to prepare a calibration curve for GFP determination (Figure 1) and a calibration curve for total protein determination (Figure 2) in leaf samples.
In the first experiment we extracted the proteins of the leaves of several the lines for each of the CO2 inducible promoters and determined their protein content and GFP expression levels. As we can see in Figure 3 not all the obtained lines were induced in high CO2.
Based in these results, we decided to carry out the second measuremet with the following line for each promoter:
- Line 2 for ADH1
- Line 7 for ATPS
- Line 9 for CYS4
- Line 1 for G3PDH
For determining the reliability of the obtained results and confirming that the selected lines are good candidates for being use as CO2 biosensor plants we decide to repeat the experiment. As observed in figure 4, all selected lines responded to high CO2. Even more, we observed that 6 hours of exposure was enough for sensing the presence of “toxic” concentrations of CO2.
Parts
Fusing visible reporter genes to selected promoters
One of our lines of work consists of fusing visible reporter genes (eYGFPuv and RUBY) to the promoters that we have previously selected. We have done this with the MultiSite Gateway cloning method (see “Protocols”)
First of all, we must produce entry clones with the promoters, eYGFPuv and RUBY into the pDONRs vectors. We did this first step in a BP reaction (using BP clonase).
Once the Entry clones were ready, we had to transfer to a secondary plasmid, the Destination vector (R4pGWB501 plasmid).
We did LR reactions (using LR clonase) to fuse, on the one hand, the selected promoters with the eYGFPuv and on the other, the promoters with the RUBY cassette. Thus we have obtained the following final plasmids:
Adding protein degradation sequence (PEST sequence)
In this line of work, we have used the Golden Braid cloning method (see “Protocols”). Using a domestication protocol to adapt basic DNA parts to the GB grammar, we introduce our PCR products, in four pUPD2, as we see in the image.
These four pUPD2 correspond to: the constitutive 35S promoter, the visible marker that we have used for this construction, which is eYGFPuv, the PEST degradation sequence and the NOS terminator.
In order to create a new transcriptional unit, we need to join all parts using a Multipartite GB assembly reaction. We used the pDGB1α plasmid as receptor.
Our final plasmid contains a constitutive promoter (p35S), that in the future would be substituted with an inducible promoter, the reporter gene eYGFPuv, the degradation sequences (PEST sequence), and the terminator NOS. To select the future modified plants, we will add the kanamycin resistance gene.
This plasmid will be used as a proof of concept to see how the PEST sequence used in protein degradation works.