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1.Source
After a long period of development, the traditional logic gates in igem comes up with a series of limits, such as a usual plasmid can not hold a big system fo its size, and when implied to another species, there are always lots of bugs, and the number of interaction between DNA and protein used as logic gate is not enough to allow us to build up a system big enough to imitate nature logic gates system.
Considering this condition, apocalyptoed by the interaction of proteins in eucells, like the G protein signal passageway, we come up with the idea that we can use items that are “of all the same” as elements in the logic passageways,for the future of our project.
2.Prototype
As is well known, RNA is considered as the start of life. Also, there are reports about using programmed DNA strings as calculation elements to compute Tic-Tac-Toe game（picture）. Thus we came up with the idea that use nucleic acid as elements, being more specific, we chose hammerhead ribozyme which tend to cut itself at special cleavage site when its activity center is of right secondary structure.（picture）
3.Inspiration
The pattern of our project was from two elite work: the paper of robert pankovsky and XXX. from the first we establish the strategy of control the activity of a ribozyme by a short RNA, while after cleavage, the short RNA resulted from cleavage can also used to modify another ribozyme, after calculation, the sequence is determined, and formed the four basic types of gates:AND, OR, NO, YES, thus form the basic pattern of our project. Another was the theory that if a ribozyme is assembled in front of a sequnce that coding protein, the coding sequence can still work if there is a spacer. However, after cleavage, for the coding sequence lost the protect in front of it, it will be digested by nucleic acid exonuclease faster and the expression of protein will decline.
4.Blueprint
a.theophylline sensor passage
The ribozyme fused with an aptamer was put in front of a GFP sequence. When theophylline is added, the cleavage is induced and the expression of protein declines, the whole RNA use a Lac promoter.
b.YES-gate
The ribozyme which lost its activity for secondary structure was set in front of a GFP, promoted by a continuous promoter, while a short RNA, the key-of-yes, was promoted by a Plac. When lac is added, the short RNA is expressed and activate the ribozyme. The cleavage is induced and the expression of protein declines
c.NO-gate
The ribozyme is activated while the short RNA, key-of-no is no added. When lac is added, the short RNA combines with the ribozyme and restains the cleavage, thus the level of GFP is increased.
d.AND-gate
The ribozyme’s activity must be activited when the short RNA key-of-and-1 and key-of-and-2 is both transcribed, induced by lac and arc.
E.OR gate
The ribozyme’s activity needed to be activited when the short RNA key-of-or-1 and key-of-or-2 is both transcribed, induced by lac and arc.
5.Future
The RNA logic gates is expected to replaces some passage ways in our logic gates. In the future, improved programmes will be developed and every lab will be able to create parts that they need. For the arrange of sequence is various, the limit of varieties of logic gates parts will be overcome and more categories of RNA elements will be developed.
