Team:NYMU-Taipei/FAQ

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Contents

General Questions

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Q. How many biobricks do you test or construct the new biobricks?
A. We have created 45 parts this year, and have tested and used many of the provided biobricks. More details about the parts we have created can be found in Parts.


Q. Could you please tell me the significance of your project? What kind of problems you want to solve?
A. We want to provide a tool that can be used in synthetic biology to reveal the intricate rules involved in biological systems. Furthermore, we have created a tool that can speed up our exploration of gene regulation in vivo.


Q. Tell me more about the rule of synthetic biology in your slides. What is the connection between rule and speedy?
A. Discovering the rules of biological system is the basis of much research in synthetic biology today. However, due to the nature of bacteria and other living organisms, finding these rules require a lot of time. Our project provides a tool that can not only help discover details about the biological world, it also works to speed up conventional research. By speeding up research, we can reveal the nature of organisms.

Q. What are the differences between a single cell and cell population?
A. When studing a cell population, we can only take the average of all of the cells expression. This way of studying an organism is synonymous with studying humans using millions as the smallest unit. However, it is often the uniquness of a specific individual that reveals the most information. Assuming that the technical aspects of single cell study can be soon overcome, we believe that to truly understand the minute details of research requires precise work on single cell research. Our project can aid this research by providing control over the cell's actions.

Q. Why we put our emphasis on a single cell? If we can get result just by a simple method in cell population, what is the need for us to use a single cell? What's the importance of using a single cell?
A. We put emphasis on a single cell simply because few have considered single cell research. Neither single cell research nor population research is better than the other. They are separate from each other; both types have advantages and disadvatages. We research on animals and humans using both population and individual research methods. This should be reflected synthetic biology. Both single cell studies and population studies should be vital components of synthetic biology.

Speedy switch

Q. What is a Riboswitch?
A. Riboswitch is a part of mRNA which can bind to a small target molecule. This reaction can then affect the activity of the gene following this riboswitch due to change in secondary structure. In essence, a riboswitch acts similarly to the promoter region of DNA and can regulate the translation of proteins from this mRNA

Q. Why do we need Riboswitches
A. Due to the fact that proteins can bind to small molecules to induce effects, riboswitches may not be essential to life. However, we can use the ability of riboswitches to bind to small molecules to regulate the genes that are downstream. Using this method, DNA is pre-transcribed, and when the genes downstream are needed, an inducer can be introduced to activate the riboswitch, thus translating the needed genes.

Q. What are the advantages of Riboswitches over Promoters
A. When promoters are used, the cell has to go through the entire process of DNA to RNA to Protein. As a result, This is a slow way to measure the reaction time of a certain experiments. For example, to measure the effect of a substance on the production of GFP after it has been introduced into the cell, we would have to wait for the cell to transcribe RNA from DNA, then GFP from RNA. If we used a riboswitch, transcription of RNA to DNA has already been accomplished. We when we introduce a substance that combines with a riboswitch, all the cell has to do is translate protein from mRNA essentially skipping the first part of the central dogma. Using this way we can speed up the reaction time of a cell towards a particular substance

Q. What are its limitations?*
A. Currently the main limitation of riboswitches is that there are only a few substances that we know can bind with riboswitches. Furthermore, sometimes RNA sequences of protein ligated to the end of the riboswitch interfere with the function of riboswitches. As a result, there are many reactions that we cannot test. However, if we can find more riboswitches and are able to pick and choose the riboswitch as well as the protein sequence that can be attached to its end, we can speed up a variety of different experiments.

Q. Why do we use GFP to test riboswitch first? What are the advantages?
A. GFP is a widely common and thoroughly tested protein. Its fluorescence makes it very easy to tell if the riboswitch is working or not. By using GFP we can first determine the viability of a particular riboswitch before we ligate other protein coding regions to the end of it.

Q. There are many different riboswitches .Why did you choose the theophylline riboswitch?
A. Although there are many different riboswitches, we needed a very specific type of riboswitch in our experiment. We had several requirements that had to be fulfilled:

  • The inducer for this riboswitch cannot be metabolized by E.coli. If the induced is metabolized, it may be digested before it has the chance to bind with the riboswitch.
  • The inducer cannot exist in naturally occuring E.coli, otherwise we would have no means of turning this switch on or off.
  • The DNA of the riboswitch cannot contain a E,X,S, or P cutting site, otherwise we would splice it accidently during transformation.

After scanning through many papers documenting riboswitches, only the theophylline riboswitch matched our descriptions.


Q. Will the untranslated mRNA with Riboswitch be degraded in the cell?
A. When a cell is in a bad environment(in our device, theophylline is toxic for E. coli.), the speed of mRNA degration is slower than under normal condition. Even though less mRNA with riboswitch is degraded, we still use a constitutive promoter to ensure that a greater amount of RNA is produced.


Q. In the whole picture, where is the riboswitch sequence located? Upstream or downstream the mRNA reporting aptamer?
A. Downstream the mRNA reporting aptamer so that we can study the movement of mRNA before inducing the riboswitch to begin translation to protein


Q. Could you please give me some references to support your theophylline riboswitch can work?
A. One of our references: Shana Topp and Justin P. Gallivan(2007)Guiding Bacteria with Small Molecules and RNA. JACS also use theophylline riboswitch as their material. Their data show that after adding theophylline into riboswitch can really work. For more reference, please check out our part: speedy switch.


Q. Could you please explain the relationship between speedy switch and single cell?
A. By using the speedy switch, we can control the flow of genetic information. We can stop and start protein translation at will. When we consider this level of control over a single cell, it becomes much easier to research how a single cell works in different enviroments, with different genetic information

Speedy reporter

Q. Why choose eIF4A as our mRNA binding protein rather than other proteins that are natively produced by bacteria ?
A. We chose eIF4A as our mRNA binding protein due to its dumbell structure. This structure makes it very simple to cleave in the middle which maintaining the interaction between the two parts of the split eIF4A structure.

Q. How can you confirm that fluorescence is emitted via GFP-eIF4A binding on the mRNA aptamer rather than random combination of the split GFP-eIF4A?
A. According to research papers, there is very low background fluorescence when the mRNA aptamer is not added into the solution. This means that the probability of random combination of split GFP-eIF4A is extremely low.

Q. It's seems you just copy the paper,what's your new idea or contribution?
A. While these ideas may not be the newest or the most innovative, they are still scarcely seen in the grand scheme of synthetic biology. SpeedyBac combines several smaller devices into a combination that can help increase the rate at which knowledge about synthetic biology is growing. We wish to bring unconspicuous ways to shorten lab times to the teams at iGEM and across the world.

Q. Plac or Ptet promoter is inducible or consitutive in your system? Why does mRNA reporting part ues two kinds of inducible promoter?
A. They are both inducible in our system. We wish to use these promoters to check mRNA reporter system works to report mRNA when they are produced.



Q. In your result assay part, why do you add IPTG?
A. IPTG is the inducer that starts production of our eIF4A aptamer.

Q. What is the time saved when using split-GFP instead of normal GFP?
A. The split form of GFP can reconstitute in the presence of RNA aptamer and begin fluorescing in about 3 mins. A normal GFP needs about 3 hours to emit its fluroscence.

Q. What is the function and feature of the linker ?
A. It is a linker between EGFP and eIF4A that does not interfere with the protein folding of either protein. Also, Glycine and Serine are used as the linker due to their flexibility.

Q. What is the difference between your method and the antibody technique?
A. Using our method, we can study the cell in vivo, rather than having to lyse the cell in order to get to the proteins.

Q. In your experiment, will the RFP and GFP FRET?
A. Conventionally mRNA and Protein is produced at the same time. This may cause the GFP that binds with mRNA and RFP that binds with protein to produce the phenomenon known as FRET. However in our experiment, we have added a riboswitch in between the mRNA level and protein levels. Using this switch we can control the flow of gene expression. We can study both mRNA and protein without having to worry about their interaction, nor the interaction of GFP and RFP.

Speedy degrader

Q. What does SsrA stand for?
A. SsrA tag is encoded by the ssrA RNA. SsrA RNA is recognized as tmRNA because it has characteristics of both tRNA and mRNA. In nature, it labels incomplete peptides, and then directs tagged proteins to several proteases

Q. There are many ways to degrade proteins. Among these methods, why choose the SsrA tag? What are the benefits?
A. Because the SsrA-SspB system is ubiquitous for eubacteria, our devices can be easily apply to other prokaryotic systems.

Q. Many scientists have already done research on the SsrA taq. What is the purpose of our experiments?
A. We want to apply the ssrA tags to different fluorescent proteins. We contribute biobricks for iGEM, providing instant fast-degraded reporters for other iGEM teams.


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