Difference between revisions of "Team:SJTU-BioX-Shanghai/Results-cn"
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<div class="figure-text"><strong>图7 抽滤结果、盒子和app的首页界面</strong> (A) 含eforRed的pET duet-1质粒导入E.coli，不同菌液浓度时使用简易装置抽滤在玻璃纤维滤纸上的效果图。 (B)为了手机拍照制作的可放置手机与目标滤纸的架子，已有成品，详见<a target="_blank" href="https://2017.igem.org/Team:SJTU-BioX-Shanghai/Detector-cn">设备</a>页面。</div> | <div class="figure-text"><strong>图7 抽滤结果、盒子和app的首页界面</strong> (A) 含eforRed的pET duet-1质粒导入E.coli，不同菌液浓度时使用简易装置抽滤在玻璃纤维滤纸上的效果图。 (B)为了手机拍照制作的可放置手机与目标滤纸的架子，已有成品，详见<a target="_blank" href="https://2017.igem.org/Team:SJTU-BioX-Shanghai/Detector-cn">设备</a>页面。</div> | ||
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| − | <div class="my-title h5-my-responsive" id="section4"> | + | <div class="my-title h5-my-responsive" id="section4">响应能力</div> |
| − | <p> | + | <p>为了验证我们所构建的STAR×色素蛋白报告系统的有效性和其响应能力，我们构建了lac操纵子介导和As金属启动子介导的两个系统。其中lac操纵子为我们所使用的质粒载体pCDF duet-1本身携带的，As启动子为iGEM2006_Edinburgh提供的part BBa_J33201。</p> |
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<img src="https://static.igem.org/mediawiki/2017/e/e1/T--SJTU-BioX-Shanghai--17yy58.png" class="img-fluid"> | <img src="https://static.igem.org/mediawiki/2017/e/e1/T--SJTU-BioX-Shanghai--17yy58.png" class="img-fluid"> | ||
| − | <div class="figure-text"><strong> | + | <div class="figure-text"><strong>图8 lac操纵子与As repressor调控STAR系统的基因回路示意图 </strong>As repressor和lac操纵子，都应插在Antisense之前，并且上图两个基因回路为target段在前，Antisense段在后相串联于同一个质粒载体中。</div> |
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<p>The characterization of lac responsiveness is mainly carried out using the STAR1 system, and sfGFP, which is more easily quantitatively monitored, is used as a downstream reporter gene to determine its accuracy. Afterwards, by using eforRed as the reporter gene, visualization of pigments was performed.</p> | <p>The characterization of lac responsiveness is mainly carried out using the STAR1 system, and sfGFP, which is more easily quantitatively monitored, is used as a downstream reporter gene to determine its accuracy. Afterwards, by using eforRed as the reporter gene, visualization of pigments was performed.</p> | ||
Revision as of 07:47, 13 December 2017
在我们的表征实验中，STAR可以有效抑制下游基因的表达。我们采用sfGFP（BBa_K515005）作为下游的报告基因，对应STAR1/3系统设计了Target有无的对照（见图1），监测其生长过程中荧光值和OD值的变化。
如图1A、1B所显示，在培养的400min里，有Target1或Target3存在时sfGFP的表达明显地降低。而在两个STAR系统的比较中，可以看出由我队设计并改进的STAR3系统的target抑制效果更好，其荧光值只略高于空白对照，与无target情况下sfGFP的表达形成鲜明对照。在实验中我们有平行进行三次生物学重复，其结果一致显示Target3的抑制下过更强。图3C表现出在培养的第600min，不同实验组的标准荧光值差距。这里我们取荧光值/OD600来消除细菌生长对荧光值带来的影响。
当系统中只有T（target）存在时，下游基因的表达被关闭；A（antisense）和T（target）同时存在时，T的抑制作用被关闭，下游基因重新活跃表达。我们以sfGFP作为报告基因，验证了STAR系统的这种功能。
图4A/4B中的结果显示在500分钟的培养中，大肠杆菌有STAR系统--Target和Antisense同时存在的情况下sfGFP的表达明显升高。图3C/3D代表考虑到OD值的标准荧光值。这些图片里的数据显示，在培养的第600分钟有STAR系统的情况下，sfGFP表达量分别升高了超过两倍（对STAR1）和四倍（对STAR3）。这说明STAR 成功地对基因回路中基因表达起了抑制作用。
为了实现可视化和同时进行多因子检测的特点，我们选择色素蛋白作为下游的表达基因。并在众多色素蛋白中，特别选用颜色区别显著的三原色RGB（red、green、blue）进行实验。我们用到eforRed(BBa_K2285012)、amilGFP（BBa_K592010）、cjBlue（BBa_K592011）、amilCP（BBa_K1357009）等色素蛋白代表对应的三原色。
在实验中，我们将Target和色素蛋白相连接，分别构建出Target 1、Target 3和四种色素蛋白相连接的系统，并在双表达载体上（STAR1系统使用pCDF duet-1载体，STAR2系统使用pET duet-1载体）同时连有对应的Antisense 1和 Antisense 3。因此，此种基因回路可供未来的使用者在Antisense前插入其对应的响应元件，如操纵子、阻遏子等，以实现不同外界刺激引起色素蛋白表达量不同的结果，进而表现为肉眼可见的色素颜色深浅不同。
| eforRed | amilGFP | amilCP | cjBlue | |
| STAR1 | In stock | In stock | In stock | In stock |
| STAR3 | Not in stock | In stock | In stock | Not in stock |
上表为我们目前已验证过的含有色素蛋白的STAR系统。我们已经提交对应的所有8种Target+色素蛋白在质粒pSB1C3上的标准化part，和对应的Antisense在pSB1C3上的标准化part。
由于人眼判断的局限性和误差，为了使我们的报告系统在可视化效果的基础上更加准确和可定量，并同时满足易操作和造价实惠的特性，我们设计了一款简易抽滤装置并配以可供手机拍照分析结果的app和为拍照分析提供稳定环境的盒子。详细信息请参考wiki的"检测"和"分析"页面。
为了验证我们所构建的STAR×色素蛋白报告系统的有效性和其响应能力，我们构建了lac操纵子介导和As金属启动子介导的两个系统。其中lac操纵子为我们所使用的质粒载体pCDF duet-1本身携带的，As启动子为iGEM2006_Edinburgh提供的part BBa_J33201。
The characterization of lac responsiveness is mainly carried out using the STAR1 system, and sfGFP, which is more easily quantitatively monitored, is used as a downstream reporter gene to determine its accuracy. Afterwards, by using eforRed as the reporter gene, visualization of pigments was performed.
From the above, fluorescence emitted by gene under the control of STAR1 system containing lac operon changes over time. The difference between the group treated with ITPG and the control becomes greater and greater. The fluorescence of ITPG group generally not higher than the control and 10 hours after treating ITPG, fold activation reach a value of 6, which indicates that our STAR system is very effective as a RNA switch. Due to the limitation of time, we haven’t finished the characterization of STAR3 and chromoproteins so we plan to continue the rest of our work in the future. What’ more, according to the results from our characterization experiment, STAR3 system has greater fold activation than STAR1. There is every reason to believe that STAR3 system can response to change of environment more accurately. STAR systems are actually competent candidates for molecular switch.
On the part of regulation of As promoter, we have built the complete STAR3 system that can sense As3+. From the electrophoretogram, the first band is about 600-700bp, indicating the existence of As+A3, and the second band, about 900bp long, indicates the existence of T3+sfGFP. All of these result prove the success of our construction. In fact, we have proved that our construct is correct via Sanger Sequencing.
Due to the strong orthogonality of STAR system and the differences between colors of selected chromoproteins, we can build a multi-factor detection reporting system. In a multi-factor detection, the double-factor detection is the most direct and easiest to distinguish. The future iGEM team can use our reporting system to build their own multifactorial detection system by adding different downstream reporter genes.
To simulate the actual situation in multifactorial responds to the color changes incurred by factors of interest, we started with co-transformation of two plasmids containing different chromoproteins.
We tried to use heavy metal ions as concrete examples of multiple factors. In the experiment, we selected As promoter and Co promoter as Antisense starting elements. Specifically, in the presence of As3 + and Co2 +, the corresponding repressor is released, activating the expression of antisense, subsequently reducing the inhibitory effect of Target. Next, downstream chromoproteins are expressed, resulting in obvious changes of colors. At the same time in a certain range of metal ion concentration, when the metal ion concentration increases, more Target are disrupted by Antisense, and the color gets brighter and brighter. And two STAR systems were established on different compatible plasmid backbone: pCDF duet-1, pET duet-1, which provided convenience for the transfer of the two expression vectors into the same host E. coli.
We have successfully construct STAR 1 system with Co promoter and STAR 3 system with As promoter separately. And the electrophoretogram and sequencing both accord with the design. But because of the time limit, we haven’t done characterization experiments about multi-factor detection.
- Chappell J, Takahashi MK, Lucks JB. 2015. Creating small transcription activating RNAs. Nat Chem Biol 11:214–220.
- 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.
