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

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.

• YES-gate

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.

• 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 restrains the cleavage, thus the level of GFP increases.

• AND-gate

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.

• OR gate

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.

• Future

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.

Recombinase

Owing to its specific DNA cutting and splicing features, recombinase have been very widely used in synthetic biology. Compared with the traditional protein regulatory pathway, recombinase can "write or erase" stable "permanent memory" in the cell, which can be passed on to future generations.

In our task, the imaging process after sensitization relies on three different protein pathways. In theory, the colors°Ø duration time depends on the illumination time and the life duration of the chromoproteins. So in order to realize the film-like "photographic memory "feature, we introduced the idea of recombinase.

In general, our thinking is like this:

The expression levels of recombinase are under the control of the upstream sensitizing system .And the recombinase will cause the inversion of certain DNA fragments, which will finally result in permanent color display. Expression of the recombinase under the control of upstream sensitizing system is easy to implement via the previous pathway design. As for the recombinase-controlled color display circuits, its design and verification have been completed in 2012 by team KAIST, which changed the expression levels of different recombinase and achieved various color expression.

[principle.png] Fig.1 The schematic of our idea. [KAIST_Experimental_Results.png] Fig.2 The experimental verification done by team KAIST. Specific colors were expressed under the control of different expression levels of recombinase.

In fact, this design makes it possible to adjust the color a single cell in. Given that the light-sensing system were replaced by another set of sensing system, the sensing signal would be converted to chromatographic output, which, compared to the traditional intensity-output system, not only make the results more intuitive, but also reduces the interference of the intensity due to environmental fluctuations.

[improvement.png] Fig.3 With the improved in-output system, the traditional output signal intensity was transformed into chromatography, which relies on the input signal and shows better anti-disturbance features.The top output bar represents the traditional output pattern, which can only presents a "deep-light" output and gets interference easily. The middle shows with three different kinds of matches of two chromoproteins, "red-green", "red-blue"& "green-blue", signals are output in chromatographies. The bottom provides expected effects of output with further improvement.

To realize the above vision completely, not only the precise modeling and analysis is needed, but also a lot of debugging work is inevitable and crucial (such as the expression level-upstream control curve, the effect of concentration on the activity of the enzyme reverse efficiency). And each time the debugging means a new plasmid construction, making it°Øs not a short-time work at all.

We have tried validating our design by experiment, but unfortunately, by the time constraint, only about two-thirds of plasmid construction work was completed.

Reference:

1. 2012 KAIST_Korea
2. Part BBa_K907000