Team:SJTU-BioX-Shanghai/Design-cn

设计

今年，我们想要创造一套可视化检测器用来检测两个或两个以上环境因子。

STAR3 系统的诞生

大家现在知道，我们称帝国理工的STAR为STAR1，而把自己构建的称为STAR3，那么想必大家会好奇为什么我们会跳过数字2，而直接命名这个新构建的系统为STAR3。接下来我们将为大家讲述STAR3系统诞生的故事。

帝国理工和我们的项目都是建立在第一篇描述STAR的文章的基础上的。 (Chappell J, Takahashi MK, Lucks JB. 2015. Creating small transcription activating RNAs. Nat Chem Biol 11:214–220.)

Figure 1. The STAR construct. In the absence of antisense, sense target RNA will form a stem-loop structure, functioning as a terminator and stop the following transcription. When antisense RNA, a mRNA fragment which is complementary to part of the target RNA, is transcribed, subsequently the terminator structure will be disrupted and then switch on the inhibited transcription.

在文章当中，共有4种分别来自于不同物种的衰减子被测试，它们作为分子开关的性能用激活倍数来表示，激活倍数就是用激活时的标准荧光值除以抑制时的标准荧光值（标准荧光值就是绝对荧光值除以OD600）。帝国理工的项目选取了性能最好的开关，来自菌株AD1。

Figure 2. Fold activation of modified attenuators. Fold activation of four modified attenuators of strain Anti-anti, T181, pbuE and AD1 respectively are labelled *. Fluorescence characterization was performed (measured in units of fluorescence/OD at 600 nm) in the absence of Antisense and the presence of Antisense in E.coli strain K12 MG1655. (Chappell J, Takahashi MK, Lucks JB. 2015. Creating small transcription activating RNAs. Nat Chem Biol 11:214–220.)

我们的目标是检测环境中的多个因子，所以我们必须要用多个STAR系统来分别控制不同的检测线路。我们注意到在原始文章中，除了AD1菌株，还有来自T181菌株的另一套STAR系统，拥有10倍以上的激活倍数，性能上仅次于AD1。因此我们把来自T181的STAR系统称为STAR2系统。四种分子开关的序列在文章中均有提供。

然而我们担心的是STAR2系统的性能并没有我们想象中的那么好，因为作为次好的STAR，文章显示也只有17倍的激活倍数，与最好的STAR1系统所显示的153倍相比，相形见绌。事实上，我们也对STAR2系统进行了测试，发现它的本底表达水平比较高而且只有很低的激活倍数。

在暑假里面，我们找到了一些其他的策略用以改进STAR2系统的性能(根据另外一篇文章 Meyer, S., Chappell, J., Sankar, S., Chew, R., and Lucks, J. B. (2016) Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies Biotechnol. Bioeng. 113, 216) 通过把J23119启动子换成更弱的常表达启动子J23150来抑制本底表达，以及通过在Antisense的5’增加hairpin结构，在3'增加scaffold结构来增强Antisense的稳定性，使其在降解之前存在的时间更长。但是我们的尝试失败了，因为改进后的Antisense2很难被插到质粒里面去，原因疑为存在太多发卡架构。不管怎样，我们还是自己想到了一个办法，从Target的角度而不是从Antisense的角度来改进我们的STAR系统，这种思路并没有在以往的文章中提及。

我们注意到尽管Target1的序列短于Target2，但它对转录的抑制效果反而比Target2要好，那么很自然的想法是，抑制本底表达可能跟终止子序列长度关系不大，关键在于序列的结构。因此，我们使用一个在线的RNA二级结构预测软件去分别预测Target1和2的结构，下面是软件得到的预测结果。(RNAstructure)

a

b

Figure 3. Structure prediction of Targets. To predict such RNA secondary structures according to DNA sequence, a online tool RNAstructure is used here. The color of each base pair indicates the probability of the structure (red indicates the greatest probability). Set Temperature to 310.15 K (37℃), Maximal Loop Size to 8, Maximum Percentage Energy Difference to 10, Window Size to 3 and Minimum Helix Length to 6. (a) Structure predicted for Target 1. (b) Structure predicted for Target 2.

基于Target2不能有效抑制转录的事实，我们想，可能两个连续的茎环结构就足以达到很好的抑制效果和激活水平。因此，通过观察Target2的序列和结构，我们决定保留Target2中第一段连续的PolyT前面的一个茎环结构，把后面的序列全部去除，作为我们设计新Target的骨架。为了让这个截短过的Target2序列保持和Target1一样的两个茎环结构，我们需要在Target2的hairpin前面再添加一个茎环结构。具体做法是，直接在序列的5’端添加碱基，使这些碱基能与下游的序列构成一个新的茎环结构。当然，为了保持Target2拥有与Target1一样的茎长度和环长度，我们在原始序列上增加了一些碱基，来保持二者的结构一致性。Target3的序列可以在 BBa_K2285020 找到。Antisense 3 的序列可以在 BBa_K2285010 找到。

Figure 4. Structure prediction of Target 1 and Target 3. Condition Setting was the same as mentioned above. The red color of base pair indicates the greatest probability. We can see that the lowest energy of T3 (Target 3) is -41.2, lower than that of T1 (Target1) which is -32.6. More red regions appear at the first loop of T3 compared to that of T1.

为了实现多因子检测，我们必须保证两套STAR系统之间互不影响，独立工作。通过之后的正交性实验，我们证明了这两套系统之间依然保持着很好的正交性。（实验数据未给出）

为了表征我们的STAR系统，我们使用sfGFP荧光蛋白作为我们的报告基因。这里是我们构建的用于表征的载体。我们选择了双表达质粒pCDF-Duet1和pET-Duet1作为我们的载体，因为这些载体有两个多克隆位点，可以同时插入我们的Target和Antisense片段。

Figure 5. Characterization of STAR 3 construct. In the absence of Antisense 3, little sfGFP will be expressed with undetectable fluorescence intensity. When Antisense 3 is transcribed, fluorescence will be emitted under ultraviolet light. Both Target 3 and Antisense 3 are under the same kind of Anderson’s promoter J23119.

Reference:

1. Chappell J, Takahashi MK, Lucks JB. 2015. Creating small transcription activating RNAs. Nat Chem Biol 11:214–220.

2. Meyer, S., Chappell, J., Sankar, S., Chew, R., and Lucks, J. B. (2016) Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies Biotechnol. Bioeng. 113, 216.

色素蛋白

下一步，我们用色素蛋白替代我们表征用的sfGFP来达到更好的可视化效果。

Figure 1. Optimized chromoprotein expression construct. In order to make our monitor system more visible and more convenient for people to use, we use chromoproteins as our reporter gene instead, which exhibit colors without ultraviolet light.

当两种不同的色素蛋白相混合时，一系列的颜色就会产生，而这些颜色会不同于原始的两种颜色，这个混合颜色可以作为判断两种因子浓度的依据。

我们相信这套系统能够在很多情况下得到应用。为了说明它的可行性，我们选择了重金属离子检测作为例子。我们构建了以下回路来检测重金属，其中cjBlue用以表明Co的存在，eforRed用以表明As的存在。它的特殊性在于我们可以仅凭一种颜色来同时反映两种因子的浓度。

Figure 2. Vector design for Co2+ detection. RcnR is a repressor for Co promoter which control the transcription of Antisense 1, then control the expression of chromoprotein cjBlue. In the absence of Co2+, RcnR binds to the Co operator region and blocks the transcription of Antisense 1. When Co2+ exists, Co2+ binds to the RcnR and inactivates it so it no longer binds to the operator. Then Antisense 1 disrupts the stem-loop of Target 1 and cjBlue is expressed.

Figure 3. Vector design for As3+ detection. AsrR is a repressor for As promoter which control the transcription of Antisense 3, then control the expression of chromoprotein eforRed. In the absence of As3+, AsrR binds to the As operator region and blocks the transcription of Antisense 3. When As3+ exists, As3+ binds to the AsrR and inactivates it so it no longer binds to the operator. Then Antisense 3 disrupts the stem-loop of Target 3 and eforRed is expressed.

small CAT

Loader

In the way of searching for a tool to load our bacteria, we have tried many possibilities. Microfluid chip is too small to vision the color, dry bacteria might be hard to control. Finally, we found glass fiber filter to act as a loader, which means to conserve and shelter the bacteria.

Glass fiber filter has the exact pore size as the E.coli. So, if we filtrate the culture solution through this filter, it will conserve the thallus and leave the nutrient liquid.

In this operation, we complete two additional purposes, condense and restrict the bacteria. When looking through the culture solution, it is hard to reckon some tiny color change because of the shallow concentration and the color of nutrient liquid. After condense onto this filter, many small color changes can be much easier be captured by camera or naked eyes. Also, we need a container to restrict and standardize the area to make it possible for analyzers to compare between different experiments.

APP

To visualize the result of the protein, we plan to take picture and transfer the result by the image and RGB value and finally get the concentration. So, the APP ColorAnalyze we designed has two main functions, taking photo samples and analyzing the color.

When sampling photos, we got two methods. First, the APP can import photos from album, so we choose the pre-cut photos to calculate the colorimetric values. The cutting operation is controlled by users, so users cut off the incorrect edge and remain the sample by subjective opinion. Another way to select samples from the photo is setting the limits. Users can set a maximum and minimum value to restrict the RGB value. We use this method to get rid of the white background and dark dots.

When analyzing the color, there are many extraneous factors need to be controlled, the most important one is the light change. We designed a box to insulate the environment, so that we can control the light consistency inside the box. Except for the box, we also develop a method to calculate the standard curve to avoid the environment interference. Users let some bacteria reacting with the solution with standard concentration, and then we can reach the concentration of the sample by searching in the standard curve.

MATLAB

This part intends to realize the function: get a standard color chart from a n*n matrix which stores color information of combinations of two kinds of inducers. In different combinations, difference of concentration would result in difference of color. We use image processing method to generate a color chart. There are a few procedures in the program.

First,Read an image.

Second,Find standard area center.

Third,Calculate standard area RGB separately.

Finally,Make a gradient color figure by linear interpolation.