Team:SJTU-BioX-Shanghai/test page 2
This year, we are trying to create a visualized monitor which can detect two or even more factors.To achieve our goal of visualization, we use chromoproteins as our reporter. Chromoproteins, containing different pigmented nonprotein groups, are known that their colors can be easily identified by naked eyes, so that we don’t need to take certain equipment with us. However, when we were doing experiment to express chromoproteins, we found leaky expression, which means exhibiting colors without inducing. We decided to use small transcription activating RNAs (STAR) to solve the problem. We add specific terminators (STAR target or Target) before RBS to stop transcription, while a small RNA (STAR antisense or Antisense) can control the target to restart transcription.
To characterize our STAR system, we use sfGFP (superfolder green fluorescent protein) as the reporter gene. Here are the plasmids we constructed. Plasmids pETDuet1 and pCDF-Duet1 are used for both of them have two multiple cloning sites (MCS), which can perfectly suit insertion of target and antisense fragments.
And by mixing two types of chromoproteins, we can create a third color with a series of different chromaticity, which can be taken as the characterization of the relative abundance of two signal molecules.
We believe our monitor can be applied in various situations. To demontrate its feasibility, we choose heavy metal ion detection as an application. We plan to use cjBlue and eforRed to indicate Co and Hg. Not only can we determine whether Co or Hg exists, but also can get their rough concentrations by analyzing mixed color. What makes it special is that we can get information about two factors at the same time only by working on one indication.
During our experiments, we found that the Star2 system didn’t function well as we expected, with leakage at a non-neglectable level. So we decided to go further to improve the STAR system according to a few strategies.
However, while basic expression in OFF state decreases, the activated expression in ON state also decreases too and we were quite worried that chromoprotein was expressed too little for us to see in naked eyes. Given above, we intended to add a hairpin to the 5’ end and a scaffold to the 3’ end of Antisense to stable it so that more Antisense will bind to target and lead to a higher level of activated expression.
Finally, we wanted to modify the STAR2 system at primary structure and secondary structure according to STAR1 system which had been proved functioned extremely well.
We noticed that although target of STAR1 (denotes Target1) is shorter than that of STAR2 (denote Target2), it still worked better. So before we stepped further, we used RNAstructure to predict the structure of both Target1 and Target2. The result is shown below.
Given the fact that Target2 couldn’t stop transcription effectively as Target1, we assumed that the two stem-loops together contributed to the high activation fold. So we retained the first stem-loop of Target2 and use the sequence upstream to make a second stem-loop, with downstream of poly T discarded. A little modification was done on order to make sure the length of the stem and length of the loop consistent with Target1. The new STAR system we design from STAR2 was denoted as STAR3 system. Noticed that STAR3 system in fact was design from part of STAR2 system, although we named it a new STAR system, it wasn’t the case when talking of its orthogonality with STAR2 system but it also had great orthogonality with STAR1 system as STAR2 system does. The prediction of Target3 using the same parameters is shown below.
