Background

Gene Pollution

Genetic pollution is the term of genetics in which the genetic information is transferred in to the organisms where it is not needed or where this information never existed before. This flow of genetic information is usually undesired and cannot be controlled. The flow of genetic information usually takes place between the genetically modified organisms into the organisms which are not genetically modified.

Genetic pollution occurs when domesticated or genetically engineered species interbreed with their wild cousins, thus polluting the wild species gene pool. It is seen as negative because it affects the wild population's evolved capability to survive, as well as spreading genes that are not found in nature.

One of the main issues of genetic pollution lies in the fact that man has tampered with the genetic structure of these species and has created a situation that would not be found naturally. Some people find no issue with genetic pollution however, stating that it is the natural course of events. One thing is certain, genetic pollution irrevocably alters a species, for better or worse.

Gene pollution doesn’t exist in theories or novels anymore. It is happening in our world. For example, “gold rice” event in China where genetically engineered rice with β-carotene was fed to children for research recently attracted people’s large attention. The serious situation calls for new way to prevent gene pollution.

Gene Encryption

In the information time, data encryption have always been a vital part of commerce, informatics and Internets. It has become a major research and investment field.

After the establishment of DNA double helix model, scientists have always been trying to store data through gene. Just as other forms of information flows, if we want to communicate through gene, we have to encrypt and decipher. Many scientists developed some other methods to encrypt in gene. For example, some researchers thought of encrypting through specific inducer. Only adding this inducer, the cell could transcript the gene and express the stored information. Here we offer one new method to encrypt through orthogonal system, and it works well.

Pollution Prevention

Biologists across the globe have proposed various solutions to conquer gene pollution, such as kill-switch. These approaches have their advantages in one way or another, but certain defects too. In this year’s project, we come up with a distinctive thinking. We want to construct a new system of orthogonal transcription-translation network, i.e. O-Key. Any genes in O-Key cannot be expressed in normal environment, and then decomposed. In this way, the O-Key can prevent the horizontal gene transfer. In our project this year, we propose several methods to construct orthogonal creature, the phages, such as T7, phi X174, and xx. What is more, we have begun to execute the plan of creating the orthogonal phi X174. Although this is just a beginning, the future of orthogonal organism and O-Key is sure to be promising.

Principles

Preventing Gene Pollution and Gene Encryption

In order to express a certain gene in an orthogonal transcription-translation system, we need both the o-ribosome and o-mRNA to form the O-Key. We are able to rationally design the SD sequence of an mRNA, to make it inscrutable to canonical ribosome. In the meantime, a plasmid manufacturing orthogonal ribosome can be transformed into the cell to help express the o-mRNA: just like a key opens a lock. This mechanism is highly effective in controlling protein expression.

What if we want to limit the expression of certain protein? What if we need to accurately regulate the expression under certain circumstances? The O-Key offers us a great choice. We can put an o-RBS to any gene that codes for hazardous protein. By controlling the on and off of the orthogonal mRNA and ribosome, we can precisely control the expression of this protein. In this way, the synthesis of dangerous proteins can be strictly restricted and controlled by O-Key, thus preventing gene pollution. The O-Key serves as a safe to contain

To take one step further OT can be applied into biological product. Say we are a pharmaceutical company, and we have a brand new bacterium that can produce an antibiotics. We want to lease bacterium to other companies, but we don't want them to resell it or spread it out. In order to protect our intellectual property, we can use a new encryption technology using the O-Key.

The RBS of the mRNA are switched to o-RBS, and we embedded the gene that manufacture orthogonal ribosome in the cells to decipher the o-RBS. However, the gene for o-ribosome needs special inducer. Thus, our company needs to constantly provide the inducer when the contract is valid. The inducer will result in o-ribosome. Together with the o-rbs, they serve as the O-Key to produce the essential protein that sustains cells' life. When the contract expires, we'll stop providing the inducer, and the bacteria will stop to manufacture the antibiotics.

As demonstrated previously, we can use the O-Key to restrict gene pollution, and construct an encryption system that protects intellectual property.

Constructing the Orthogonal Phages

The reason why we choose phages are mainly based on its simple replication process and relatively small genome. At first, we wanted to build a fully orthogonal cells. But minimal genome for a cell is more than 300, which is obviously beyond our abilities. So we chose smaller genome creatures, the phage. As different phages have different genome. For the large

We could apply the O-Key to one particular regulation. Why can't we apply it to the entire organism? Furthermore, bacteriophage is one way of gene transmission. For the phages with large genome(more than 20kb), we could divide the whole genome into several little parts, mutate one by one genome and then assembly with "Assemblyer in Yeast", for the phages with small genome, we could mutate directly one by one or firstly divide the genome into small parts and then assembly in Gibson assembly method. As the mutation methods are all site specific mutations, we just take phi X174 for example.

We will mutate the genome one by one. Once we mutate one gene, we will transform the mutated phage DNA into orthogonal and normal cells to check whether the phage could still replicate, which is revealed by phage plaques. For the overlap gene in the phage DNA, if mutated RBS does affect other genes, we will put this gene before gene A and close the original gene by changing its Start codon. For larger genome, we just need to divide the genome, mutate one by one and then assembly in “Assembler in Yeast”, for more information on "Assembler in Yeast".

For the limited time, we just plan to construct fully orthogonal phi X174. Why we choose Phi x174 are based on following facts.

1. The first creature that was sequenced whole genome;
2. The second artificially synthesized viral;
3. We have knew clearly about it(if searching in goggle scholar, articles>300);
4. The replication process is simple and clear.
5. The genome is small, containing only 11 gene.
6. Phi X174 will not affect the common E.coli.

Experiment

Ampr RBS Mutation

We used the orthogonal principle described in the previous section to mutate the Ampr RBS, and constructed an orthogonal transcription-translation system that is independent of the existing system.

The SD sequence of Ampr RBS is XXXXXX, and we rationally designed the orthogonal sequence to be XXXXXX. We further mutated the Ampr RBS of the RFP operon, and transformed plasmid into E coli.

Here, we chose two distinct competent cell: the normal competent cells; the orthogonal competent cells containing o-16S rRNA. Plate the cells on Amp LB plates. As we can predict, the normal competent cells would not be able to express the Amp resistance protein, thus cannot survive. On the contrary, the orthogonal cells can resist Amp in the plate and express the RFP.

Process

1. Using PCR to mutate 16sRNA on the P15a plasmid to get the mutated P15a with O-16sRNA.
2. Changing RBS of Amp r gene on PBR322 to O-RBS.
3. Cotransfomating the above two mutated plasmids into cells， plating Amp LB plates, cultivating for 12-24hours.

Both of plasmid used above contain RFP gene to display the results and Pbad promoter induced by Ala; only if Ampr gene could be expressed, cell will reproduce, RFP gene be expressed and the colony turned red.

Results

From the two flats, we could easily find that the left plant plated without Ala，which means cells only containing normal ribosome. There is no colony on the plate. The reason is that without no orthogonal ribosome, Amp r gene could not be transcripted and translated，then cells could not survive on LB flat with Amp. On the other hand, the right flat, added Ala, containing orthogonal ribosome, have red colonies. Existing orthogonal ribosome means that cells could transcript and translate the Amp r gene, survive in Amp situation, the RFP could also be expressed with the help of normal ribosome.

Plan of Phi-x174

When dealing with phage experiment, many teams will face a difficulty when the phage infect other bacteria.

As for the limited time, we just do some Preliminary experiments and design the primers needed for latter work.

Model

Prospect