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Team:NYMU-Taipei/FAQ
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Contents |
General Questions
Q. What is this F.A.Q all about?
A. It's all about answering questions people might have.
Q. Why did the chicken cross the road?
A. To get to the other side.
Q. Why did the dinosaur cross the road?
A. Because chickens weren't invented.
Q. Why SpeedyBac? How fast to do what?
A. .
Team:NYMU-Taipei/Experiments#Parts.">
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 Team:NYMU-Taipei/Experiments#Parts.
Q. Could you please tell me the significance of your project? What kind of problems you want to solve?
A. We provide a tool for synthetic biology in order to reveal the rules in biological system. For understanding the gene regulation in vivo quickly, our speedyBac can serve this purpose.
Q. Tell me more about the rule of synthetic biology in your slides. What is the connection between rule and speedy?
A. .
Q. What are the differences between a single cell and cell population?
A. A single cell has its unique gene expression; while cell popution has a overall gene expresion, which means that cells in a population may have difference gene expressions from each other.
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. On the time aspect, we will want to do a more specific assay(specific result) rather than the average assay(result of average). The biological clock and growth cycle
of every cell are different, just like we Homo Sapiens having the same genome but totally versatile from one person to another.
Riboswitch
Q. What is a Riboswitch?
A. 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's the adventage?
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. Will the untranslated mRNA with Riboswitch be degraded in the cell?
A.
Q. In your whole picture, where will the riboswitch sequece locate? Up or downstream the mRNA reporting aptamer?
A. Downstream the mRNA reporting aptamer.
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. As the project mentioned,speedy switch is the critical switch between mRNA and protein.
mRNA Binding
Q. Why choose eIF4A as our mRNA binding protein rather than other proteins that are natively produced by bacteria ?
A.
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 the paper,they did this experiment without aptamer. And the result was no any fluorescence!
Q. How much time can we save to show promoter activity than traditional methods?
A.
Q. Plac or Ptet promoter is inducible or consitutive in your system? Why mRNA reporting part ues two kinds of inducible promoter?
A.
Q. Could you please tell me what is mRNA reprting part different from the conventional way?
A.
Q. In your whole project, how can you measure fluorescence from mRNA binding part? The single cell will move, so how can you make sure the single cell you meassure is the same?
A.
Q. In your result assay part, why do you add IPTG?
A.
Q. Could you please tell me the degree of fluorescence decline between splt-GFP and normal GFP?
A.
Q. In mRNA repoting part, will the fluorescence be a single pulse if you just focus on single cell?
A.
Q. Will eIF4A be fluenced by the other eIF system in bacteria? Can this system be applied to the Eukaria cell?
A.
Q. Could you please tell me the function and feature of the linker ?
A.
Q. What is the difference between your method and the antibody technique?
A.
Q. In your experiment, will the RFP and GFP FRET?
A. Although we know transcription and translation will proceed at the same time, we provide a idea that is to insert a riboswitch between the aptamer part and adapter part! When the aptamer is bound to our EGFP-eIF4A system, it will make the EGFP reconstitute a functional fluorescent protein. So, we can view green fluorescence. When you want to have a target protein expression, you can turn on our speedy switch by add a inducer to it. It will make red fluorescence.
SsrA
Q. What does SsrA stand for?
A. Small stable RNA A, a name used for tmRNA
Q. There are many ways to degrade proteins. Among these methods, why choose the SsrA tag? What are the benefits?
A.
Q. Many scientists have already done research on the SsrA taq. What is the purpose of our experiments
A. We want to construct a biobrick for iGem and help the future team to do fast degradation.
Q. Why do we choose LVA tag for degradation among tags with the same function?
A. Previous studies suggest LVA tag has been the most efficient tag to shorten the half-life of tagged protein in Escherichia coli.
Q. Why you construct so many different kinds of fluorescent proteins?
A. So we can use it in other parts of the project, for example, in the Speedy reporter part.
Q. We can choose the tag we want, however, why we need to make SSPB?
A.
