Team:SJTU-BioX-Shanghai/Results-cn

结果

抑制泄漏

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。

图1 抑制泄漏表征质粒的示意图 其中含Target1的表征质粒构建在pCDF duet-1质粒上，含Target3的表征质粒构建在pET duet-1质粒上。这两种表征系统都配以常启动子J23119，和同样的终止子。

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。

图2 Target在大肠杆菌DH5α中的表征实验（A）有或无Target1的大肠杆菌标准荧光值随时间变化曲线（B）有或无Target3的大肠杆菌标准荧光值随时间变化曲线（C）培养结束点上无Target和有Target1、Target3的大肠杆菌的标准荧光值柱状图我们使用两种在设计部分介绍过的质粒来做STAR系统的表征。不含Target的实验组中转入的质粒不含Target序列而只存在常启动子J23119。标准化荧光值是用培养菌液的荧光值除以OD600。背景对照组是使用没有转入质粒的大肠杆菌DH5α。误差棒代表三次重复的标准差。

分子开关

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3 STAR作为分子开关表征质粒示意图，其中含STAR 1的表达系统构建在pCDF duet-1质粒上，含STAR 3的表达系统构建在pET duet-1质粒上。每种STAR系统都是分别构建在一个质粒上的。

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。

图4 STAR系统在大肠杆菌DH5α中的表征实验。（A）有无Antisense1表达的含STAR1系统的大肠杆菌标准荧光值随时间变化曲线。（B）有无Antisense3表达的含STAR3系统的大肠杆菌标准荧光值随时间变化曲线。（C）终点（第600min）STAR1系统有无Antisense1表达的标准荧光值柱状图。（D）终点（第600min）STAR3系统有无Antisense3表达的标准荧光值柱状图。我们使用在实验设计部分介绍过的单个质粒作为STAR系统表征实验的载体。没有STAR的实验组转入的质粒中不含STAR系统的序列。标准化荧光值是用菌液的荧光值除以OD600。背景对照组是使用没有转入质粒的大肠杆菌DH5α。误差棒代表三次重复的标准差。

STAR系统×色素蛋白

In order to achieve visualization and multi-factor detection simultaneously, we selected chromoproteins as downstream expression gene. In a large number of pigment proteins, we chose those with obvious color distinction, that is the three primary colors RGB (red, green, blue) for the experiment. We used eforRed (BBa_K2285012), amilGFP (BBa_K592010), cjBlue (BBa_K592011), amilCP (BBa_K1357009) to represent the corresponding three primary colors.

In the experiment, we linked Targets with four chromoproteins. On the dual-expression vector (STAR1 system on pCDFDuet-1 vector, STAR2 system on pETDuet-1 vector) and the relevant Antisense 1 and Antisense 3. Therefore, for future users, this kind of gene circuit can be inserted downstream of other corresponding response elements, such as operon, repressor and so on to obtain different expression of chromoproteins induced by different external stimuli, and then the visible depth of different colors can reflect the quantity of the stimuli.

Figure 5 four kinds of chromoproteins we used in the construction of expression vector.

Figure 6 T + chromoprotein + A gene circuit diagram. In the project we ligated two genes in the same expression vector, so transform one plasmid can achieve to report a factor. In this way, not only the burden of the host can be reduced, but the multi-factor detection will be convenient as well.

	eforRed	amilGFP	amilCP	cjBlue
STAR1	In stock	In stock	In stock	In stock
STAR3	Not in stock	In stock	In stock	Not in stock

The table above shows the STAR system with chromoproteins that we have now verified. We have submitted standardized parts of the corresponding all eight Target + chromoprotein on the plasmid pSB1C3, and the corresponding Antisense on pSB1C3. We also welcome the future iGEM teams to contact us and we are happy to provide a complete plasmid for the STAR 1 / STAR 3 × chromoproteins on the dual-expression vector (pETDuet-1, pCDFDuet-1) for further construct other circuit using for detection.

In order to prevent the limitations and errors produced by human eyes and achieve greater accuracy and convenience to measure on the basis of the visualization effect, and at the same time to meet the easy operation and cost-effective characteristics, we designed a simple pumping device, an app for analysis of results and a box providing a stable environment for the camera. More detailed information please look to the Detector and Analysis page of our wiki.

Figure 7 the result of filtration and box and app front page (A) pET duet-1 plasmid with eforRed gene was transformed into BL21(DH3) E. coli, and cells with different concentration were filtered on the glass fiber filter. (B) the schematic map of the box which can give a proper distance between mobile phone and filter to be photographed. We already have a finished product of this box, to see more information please click the Detector page.

Response ability

During experiments to validate the responsiveness of the STAR system, we constructed two systems mediated by lac operon and As promoter. The lac operon is the one on MCS1 of plasmid pCDFDuet-1, and As promoter is provided by iGEM2006_Edinburgh BBa_J33201.

Figure 8 lac operon and As promoter control STAR gene loop diagram. As promoter and lac operon should be added on the upstream of Antisense, and the two circuits is constructed on the same plasmid.

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.

Figure 9 Characterization of lac operon and As promoter for STAR. A) The presence of IPTG-induced STAR 1 system with lac operon in the case of fluorescence versus time. The plasmid we use is pCDF duet-1. Characterization cells are BL21 (DH3) E. coli. And each control group and experimental group were formed from the same colony. And a total of three repeated tests were conducted to verify the effectiveness. B) with eforRed as the downstream reporter gene STAR 1 system under lac operon’s manipulation of the color characterization C) As + STAR 3 system enzyme electrophoresis diagram. The marker used in the figure was DL2000, and the third lane was an expression product constructed using the restriction endonucleases NdeI and KpnI digested at both ends of As + antisense 3, and the second lane was a restriction on the use of T3 + sfGFP Endonuclease PstI and EcoRI digested to construct the expressed product. The electrophoresis tank buffer was 0.5 x TBE and the gel contained 1% agarose, using Dye DuRed, running 120 v / 100 mA for 20 min.

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.

Multi-factor detection

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.

Figure 10 Characterization of chromoprotein co-transformation.

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.

Figure 11 gene circuit of multi-factor detection with STAR

Figure 12 electrophoretogram of Co+STAR1、As+STAR3

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.

Reference

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.