Our early brainstorming sessions were very effective and resulted in countless ideas regarding our project idea and scope. After several meetings and debates we narrowed it down to 3 main topics: pathogenic bacteria detecting sensor for food, possible upgrade for probiotic bacteria or using CRISPR to introduce information to bacteria genome as a binary code. From those, the CRISPR project was the one we initially chose. However, during the research phase, we found an issue that can not be solved within a reasonable timeframe and we had to develop a new idea. That is how our current project came into fruition.
In a world where computers are getting increasingly life-like, it is our goal to introduce computational logic in living organisms. As NTNU’s contribution to the 2016 international iGEM competition, we will make a system for introducing logic gates in living cells. Computers work by moving information between computational units, called gates. Each of these gates exhibits a simple logical statement (AND, OR, NOT...). The implementation of these logic gates is the foundation for all of the life-changing applications computer technology has provided. Our goal is to bring the great potential of computer technology, such as developing a new generation of biosensors, improving the process of industrial fermentations, or advancing cancer research to the field of biological engineering. In order to achieve this, we are making a system for engineering in vivo logic gates. We are planning to introduce XOR logic gate into E.Coli genetic code.
As in computer technology, the possible applications of introducing logic gates in living cells are, seemingly, only limited by imagination and skill. We believe that the introduction of logic gates in living cells will have applications in fields ranging from medicine to environmental science and industrial biotechnology. Some relevant applications are:
- Whole cell biosensors that can detect many different components in a predefined combination
- Tighter, and more complex, control of metabolism during industrial fermentations
- Cells that can detect multiple cancer markers and self-destruct if a given number of markers is present.
Living cells are basically computers whose task is to create more of themselves, and they are very good at it. However, it is difficult to make them do any other complex task. The reason is that proteins are an essential part of the cell's control-system and they are almost impossible to engineer. Short pieces of DNA have been demonstrated to perform complex logic tasks in test tubes and are much easier to engineer. Unfortunately, living cells do not produce them by default. However, some viruses do, and when they infect cells they sometimes leave behind the machinery that they use to make short DNAs. These machines are called replicons and the short pieces of DNA that they produce are called msDNA. We will use short DNA logic inside living cells and show that it works. We will use replicons and msDNA to accomplish this.
The msDNA based logic that we are using is based on the ability of certain DNAs, called deoxyribozymes to cleave DNAs and RNAs at defined positions. In the picture we can see a DNA nuclease (red) bound to an RNA molecule (green). Using complementarity (straight sections) to find a specific locus on the RNA molecule, the nuclease binds to it and cleaves it, using a catalytic loop (red capital) next to a G nucleotide (green capital). See the figure to the right.
We have created scripts to design our logic gates. The scripts have been uploaded to Github in the spirit of iGEM's commitment to knowledge sharing. You may access the scripts here.
Our approach is thus based on retrons producing msDNAs that cleave RNA and DNA. For our proof of concept we are making an XOR logic gate. XOR gates are essential for computer addition. They are also very rare and difficult to create using standard biological tools.What we have achieved so far:
Scheme below (click for higher resolution) shows particular steps of our experimental work. Currently, we are modifying DH5α and ER2925 E.Coli cells as it is shown in Case 1 .
Improving Other Projects
In addition to our own work, we have strived to improve previous biobricks/projects in iGEM. We charatcerized and improved the CI repressor biobrick found here.