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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 that cannot hold a big system of its size, and when applied 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 natural logic gates system.
Considering this condition, apocalyptoed by the interactions 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 future of our project.
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 specificly, we chose hammerhead ribozyme which tends to cut itself at special cleavage site when its activity center is of right secondary structure.（picture）
Inspiration
The pattern of our project was from two elite works: the paper of Robert Pankovsky. From the first one we established the strategy of controling the activity of a ribozyme by a short RNA. After cleavage, the short RNA resulted from cleavage can also be used to modify another ribozyme. After calculation, the sequence is determined,and four basic types of gates(AND, OR, NO, YES) are formed, thus forming the basic pattern of our project. Another is the theory that if a ribozyme is assembled in front of a sequence that codes protein, the coding sequence can still work if there is a spacer. However, after cleavage, for the coding sequence lost the protection in front of it, it will be digested by nucleic acid exonuclease faster and the expression of protein will decline.
Blueprint
- theophylline sensor passage
- YES-gate
- NO-gate
- AND-gate
- OR gate
- Future
The ribozyme fused with an aptamer is put in front of a GFP sequence. When theophylline is added, the cleavage is induced and the expression of protein declines. The whole RNA uses a Lac promoter.
The ribozyme which loses its activity for secondary structure is set in front of a GFP, promoted by a continuous promoter, while a short RNA, the key of yes, is 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.
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 restrains the cleavage, thus the level of GFP increases.
The ribozyme’s activity must be activated when the short RNA key of and_1 and key of and_2 are both transcribed, induced by lac and arc.
The ribozyme’s activity needs to be activated when the short RNA key of or_1 and key of or_2 are both transcribed, induced by lac and arc.
The RNA logic gates are 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 arrangement of the sequence is varying, the limit of varieties of logic gates parts will be overcome and more categories of RNA elements will be developed.
