First we organize a cold regulation system at post-translational level using a constitutive gene that E. coli has in order to cope with a sudden downshift (from higher temperatures down to 15ºC -10ºC). Also we had added an antifreeze protein, in order to help the cell to prevent water from undergoing a freezing process creating strong hydrogen bounds and inducing a vitrification process rather than a normal crystalization process. In that way the cytoplasm and the cell membrane are not damaged at freezing temperatures. Both strategies may suggest the assembly of new biobricks that can function as a “mini cold kit” to both synthesize proteins and build an E. coli mutant that viable at low temperatures. We are aware of commercial freeze induced vectors such as the P. cold, however, thess vectors are patented. As we are trying to have a fully non-patented assemblage in the IGEM community, we want to compare tese vectors with ours. In that way our "mini cold kit" would be available for anyone. We are also trying to overcome other human issues like the plants and crops maintenance, in order to survive cold weather countries like Canada, USA, Northern European countries and of course the frosts occurring in the north of Mexico and other high regions of country. In order to improve the efficiency of our cold system we build four structures to prove the different kinds of regulation that the mechanism can allow.
• First we took the promoter with all the parts involved, from the promoter polymerase-binding zone, to the Downstream Box that is inside the protein DNA sequence.
• In a second construction we took all the regulation system except the promoter to define if another promoter different to the CspA can be regulated with the freeze system.
• We also have the CspA promoter alone so we can put it controlling another gene and see if this can function at low temperatures.
• For a final construction, we took the Cold Box, the UTR region and the Shine-Dalgarno sequence with the exception of the Downstream Box and the promoter. This one will help us to compare the expression of the proteins at low temperatures without the influence of the Downstream Box effect. We suspect that the proteins will have a lower expression rate than the one we could have from the second construction.
For AFP we took fourteen sequences of the genus Zoarces americanus and make a multiple alignment. Of this we obtain a consensus sequence to choose from preferential codons of E. coli. This final sequence was sent to synthesize with no promoter in. We tried to construct the "mini cold kit" with the regulatory systems and the AFP just in front of them so we can express it with the cold shock inducible settings. In that way we ensure that the expression of the protein is controlled by the cold shock downshift. Other problematic we are overcoming regarding this mini cold kit is that the AFP's denatured at physiological temperatures. This is a problem if we want to express the protein at high levels. So when we take the regulation system, which works better at low temperatures, we are avoiding the denaturing problem when the translation occurs. The regulatory system maintains the cell genetic machinery working well so we can express the protein and produce a lot of it.
Machinery that regulates the expression + AFP = "E.coldi"
This is our final construction and is the one we are expecting to serve as an antifreeze system.
When we have many bacteria producing the AFP, the final objective is to purify the protein and to put it above the crops to see if it can prevent the formation of frost on the leaves.
If the AFP works well the frost will not be formed and the plant may carry out gas exchange so it will live much longer.
So what we choose to use this protein?
We saw many advantages for the future applications:
- The AFP is from a vertebrate protein and it could be more compatible in transplants.
- Its size is 60 aa.
- Its desnaturalization is the physiologic temperature (37°C) and don’t leave traces on tissues when the temperature shifts up
- Its located inside and outside the cells, so it is possible spray protein in plants
- Its structure is known and can be compared on the databases.
We consider that this protein could be a solution in the future Mexican crops and medicine.