Team:BHSF/Engineering
Engineering success
Our project is based on the engineering design of genetic circuit. To validate such a complex system as we designed, we separated the genetic circuit into different parts to verify if the parts are successfully operating, step by step. After getting sure each parts working normally, we would integrate the parts to see if they can work together.
Toggle switch
First of all, one of the key elements in our genetic circuit is a toggle switch. Toggle switch consists of two promoters that are placed in different directions with their downstream genes. Each side of the downstream genes can be translated into protein can inhibit the promoter on the opposite side. As a result, under natural conditions, only one of the two genes gets to express, stability. Such a system can be artificially adjusted by adding the product protein of the repressed side, it will trigger the inhibition of the other protein and turn the switch to an opposite steady state, which we call On or Off. Since the toggle switch are normally constructed in bacteria, we changed the promoters into eukaryotic promoters and transferred the circuit from prokaryotic cells to eukaryotic cells to equipt the yeast with toggle switch.
In order to test whether the toggle switch can operate normally in eukaryotic cells, we first separated the toggle switch into two parts, each of which consists only one promoter and a reporter. The promoter is a modified constitutive promoter with an operator sequence inserted downstream of its TATA box. The repressor protein can therefore bind to the operator and suppress expression of the reporter gene downstream.
We will use fluorescent protein as the reporter to verify if the promoter can be successfully repressed. There will be two groups in the verification experiment of the promoter: experimental group and control group. The experimental group would contain the galactose promoter which can start the expression of inhibitor protein and cause the green light in the product protein weakened or disappear when galactose is added to induce the expression of the inhibitor; while the control group which does not contain inhibitor gene controlled by galactose promoter would not show the same change. After induction, the experimental and control groups are expected to represent different levels of fluorescence, and therefore, the on and off of the promoter. If it turns out as expected, which is the weakened green light in the experimental group and the normal green light in the control group, it means the promoter is operating as expected in the yeast.
Since the single promoter can function properly, we then integrate two promoters of such kind to form the two-sided toggle switch. In the toggle switch, for example, exists Promoter A and Promoter B, expressing Protein A and Protein B, respectively. The Protein A inhibits Protein B, and vice versa. This structure provides the switch a stable and fixed pathway that guarantees the gene is expressed in a single direction. In order to verify the validity of the toggle switch, we again added the genes of fluorescent proteins to each side downstream of promoter A and promoter B. Fluorescent protein that gives out red light with Promoter A and fluorescent protein that gives out green light with Promoter B. When Promoter A is active in the pathway, it promotes the expression of Protein A and inhibits the expression of Protein B at the same time. Then a stable red light and a weakened green light can be observed at macro level. Similarly, when Promoter B is turned on in the pathway, it promotes the expression of Protein B and inhibits the expression of Protein A at the same time. Then the stable green light and the weakened red light can be observed. If the result turns out as expected, it indicates the toggle switch is successfully built in the yeast.
Integrase
In the verification of the integrase to reverse DNA sequence, we put a constitutive promoter in front of an inverted sequence of fluorescent protein. On either side of the inversed sequence we added the recognition sites of integrase, expecting the integrase to inverse the sequence. After the alignment of the fluorescent protein to the constitutive promoter, fluorescent protein would be produced, glowing green light. If the yeast does not give out green light before the expression of integrase and gives out green light after the expression of integrase, the integrase can successfully inverse the sequence.
Galactose Promoter
The reason why we didn’t choose to use a inducible promoter to directly control the expression of the inhibitor is that the functioning time of galactose is limited. Galactose would run out after adding it into the system and the promoter will therefore function for a specific period of time. Under that condition, we would have to artificially add galactose to the system periodically to maintain the expression of genes. This may lead to a change in flavor of the bread. In order to solve the problem, we combined galactose promoter with integrase to make a toggle switch function properly in our timer yeast. The promoter sequence before the inhibitor gene is inversed, making the polymerase unable to start transcription. Galactose would trigger the promoter to express the integrase, then integrase would turn the promoter sequence over and start transcription. This would be a stable system that does not require extended addition of galactose.
Integrated System
In order to verify the final system, we again added the gene of fluorescent protein to the downstream of each promoter in the toggle switch. Expectation would be: the yeast gives out green light when galactose is added to the system and gives out no green light when galactose is absent. Finally, the whole system is successfully built and verified step by step, ensuring each element is operating well.
Results
