1.Introduction

As shown in the enzyme kinetic equations,enzyme concentration plays an important role,as well as substrate concentration,in enzymatic reactions.In order to enhance the efficiency of an artificial metabolic pathway with a predictable substrate concentration,the expression of each enzyme in this pathway should be carefully considered.Overproduction of an enzyme may cause an excessive burden for the cell.On the other hand,insufficient protein expression can result in low efficiency of the metabolic flux.Thus,the promoter,which in fluences the absolute protein expression quantity of the enzyme,should be chosen appropriately.However,a method of measuring the absolute protein expression quantity of a specific promoter has not yet been reported.The characterization of a promoter is traditionally performed by measuring the fluorescent intensity or RT-PCR results[1-6]of the downstream protein or gene,which can only be compared in a relative manner.However,certain challenges affect the measurement of the absolute protein expression quantity of a specific promoter.The dynamics of gene expression are in fluenced by the protein coding sequences;this means that measuring a promoter by simply detecting its downstream protein expression level is not accurate[7].In addition,it is inconvenient and expensive to measure the absolute protein expression quantity using western blotting,ELISA,or similar techniques.

心之所向，素履以往。生如逆旅，一苇以航。陈老师是平凡的追梦者，也是不凡的造梦者。他总是用充满期待的爱，滋润“路途劳累”的莘莘学子。正如德国哲学家雅斯贝尔斯说过：教育意味着一棵树摇动另一棵树，一朵云推动另一朵云，一个灵魂唤醒另一个灵魂。

RiboJ is a ribozyme that self cleaves,thereby removing the upstream region and,most importantly,the 5′untranslated region.In the work of Lou et al.[7],the relative expression levels of two different variants of green fluorescence protein(GFP)(i.e.,sfGFP and cl-sfGFP fusion)were not the same.However,when they added RiboJ before the coding region,the relative expression levels for the induction curves collapsed.This finding indicated that RiboJ can act as an insulator to homogenize the relative protein expression level for a given promoter,thereby making it possible to evaluate the protein expression ability of a specific promoter on a large scale,regardless of the coding sequence.To characterize the promoter in a quick and simple way,we attempted to determine the relationship between absolute protein quantity and enhanced green fluorescence protein(eGFP) fluorescence by making an eGFP fluorescence standard curve,and then using a protein expression system with a RiboJ design to determine the promoter strength.We chose nine promoters from the Anderson promoter family,which is anσ70 transcriptional promoter library of Escherichia coli(E.coli).The Anderson promoter family is widely used in the International Genetically Engineered Machine(iGEM)Competition,and has been characterized by expressing red fluorescence protein(RFP)and GFP[4,8].Our design allowed us to successfully measure these nine promoters,and yielded higher-accuracy data for future pathway design.Our method also provides a straightforward way to standardize the strength of different promoters.Thus,this paper introduces a novel method to measure the absolute protein expression quantity of any constitutive promoter.

采用Diener等人编制的心盛量表(Flourishing Scale, FS)来测量机能幸福感，该量表共有8个项目，用于评估社会-心理机能的核心方面，在中国被试中有较好的信效度(Tang, Duan, Wang & Liu, 2016)，采用李克特7级计分，分数越高表明心盛水平越高，本研究量表的Cronbach’s α系数为0.89。

2.Materials and methods

2.1.Plasmids construction

The pET28a-eGFP plasmid was obtained from the State Key Laboratory of Biotherapy,in China.The eGFP gene was originally obtained from plasmid pEGFP-N1,and was then ligated into pET28a vector with a 6×His tag for purification.The measurement plasmid originated from BBa_J364001 in the pSB1C3 vector from the Registry of Standard Biological Parts.RiboJ was generated from synthetic oligos(TsingKe Biological Technology Co.,Ltd.,China)and was seamlessly added to the downstream of Anderson promoter sequence by means of Gibson assembly.The GFP was then replaced by eGFP by means of Gibson assembly in order to construct the final measurement device with a J23106 promoter.The other measurement devices,with different promoters,were generated by oligonucleotide-directed mutagenesis(KOD-Plus-Neo,TOYOBO Co.,Ltd.,Japan;DpnI,New England Biolabs Inc.,USA).The sequences for the Anderson promoters are documented in the Registry of Standard Biological Parts[8].The plasmids used in the study are listed in Table 1.The gene structure of the measurement device is shown in Fig.1,and the sequence is documented in the Appendix A(Table S1).

1.1.1 纳入标准 ①女性；②术前粗针活检病理证实为浸润性乳腺癌；③病灶≤5 cm，临床及影像学检查提示腋窝淋巴结阴性；④患者选择乳房全切，术中冰冻病理提示SLN有宏转移且进一步行ALND；⑤术后常规病理证实SLN有转移以排除术中冰冻病理假阳性。

2.2.Purification of the eGFP

The expressing plasmid(pET28a-eGFP)was transformed into E.coli BL21(DE3)cells.Transformed cells were grown in 1 L lysogeny broth(LB)supplemented with 34 μg·mL-1 kanamycin at 37 °C,with agitation at 220 r·min-1 overnight.When the OD600(optical density measured at a wavelength of 600 nm)reached 0.6-0.8,the cells were induced with a final concentration of 0.5 mmol·L-1 isopropyl β-D-1-thiogalactopyranoside(IPTG).After being cultured for a further 18 h at 16°C,the cells were harvestedby centrifugation at 4 °C and 4000 r·min-1 for 10 min.The harvested cells were suspended in 40 mL lysis buffer(PBS buffer,140 mmol·L-1 NaCl,2.7 mmol·L-1 KCl,10 mmol·L-1 Na2HPO4,and 1.8 mmol·L-1 KH2PO4,at pH 7.5)and lysed by a high-pressure homogenization instrument(JN-02C,JNBIO Co.,Ltd.,China).The lysate was then subjected to centrifugation(4 °C,15 000 r·min-1,30 min;Thermo F21-8x50y rotor).The clear supernatant was collected and loaded onto a nickel-nitrilotriacetic acid(Ni-NTA)column pre-equilibrated with the lysis buffer at 4°C,and the pellets were discarded.The column was washed three times with 3-5 column volume(CV)of washing buffer(10 mmol·L-1 imidazole dissolved in PBS buffer)and three times with 3-5 CV of elution buffer(200 mmol·L-1 imidazole dissolved in PBS buffer).The eluted protein containing eGFP was concentrated in a 15 mL concentrator(Amicon®Ultra-15,Merk Millipore,USA)before being loaded onto the Superdex 75 10/300 GL(GE Healthcare Conglomerate,USA).The single peak of eGFP was collected,and the eGFP was concentrated to 25 mg·mL-1.Part of the protein was analyzed immediately;the remaining portion was frozen using liquid nitrogen and kept in stock at-80°C.We repeated the purification method five times and obtained five batches of purified eGFP for further measurement.

Table 1 Plasmids used in this study.

Plasmid name Description pJ23100 Measurement device containing promoter J23100 pJ23106 Measurement device containing promoter J23106 pJ23107 Measurement device containing promoter J23107 pJ23108 Measurement device containing promoter J23108 pJ23109 Measurement device containing promoter J23109 pJ23113 Measurement device containing promoter J23113 pJ23114 Measurement device containing promoter J23114 pJ23117 Measurement device containing promoter J23117 pET28aeGFP eGFP gene inserted into a pET28a plasmid for protein purification

2.3.eGFP stability test

Next,we collected all the data from the fluorescent quenching experiment,as shown in Fig.4.The data collected by continuous measurement over 16 d was analyzed by linear regression.The results showed that the fluorescence intensity of the eGFP and the absolute protein concentration show a strong linear correlation(r2=0.9936);this means that the purified eGFP has a level of high stability and can resist quenching over time,because the measurement results from each time were similar and formed a linear correlation.

The first batch of purified eGFP was stored at-80°C;we then repeatedly measured the fluorescence intensity and absolute protein concentration 4,8,12,and 16 d after purification in order to test the stability of the eGFP and identify the impact of fluorescence quenching over time for our experiment.

2.4.Cell density and fluorescence measurement

The reaction rates of these two reactions are represented by v1 and v2,and the concentrations of compound A,B,and C are ［ A］,［B］,and ［ C］.

Fig.1.Gene structure of the measurement device.The diagram above portrays the measurement device gene structure,in which there is no sequence between the Anderson promoter and the insulator RiboJ.Part of the gene sequence is shown below,colored to align with the elements above.B0034 is a ribosome binding site and B0015 is a double terminator;their description and sequence are documented in the Registry of Standard Biological Parts.

2.5.Modeling and data analysis

This section first discusses the theory of protein production relative to promoter activity[10,11].The velocity of the production per cell of eGFP mRNA molecules(M eGFP)is relative to the copy number(n),promoter activity(PoPs),and mRNA degradation rate(γMeGFP),as described in Eq.(1).The amount of immature eGFP per cell(I eGFP)is dependent on the immature eGFP translation rate(),mature eGFP degradation rate(γIeGFP),and mature eGFP translation rate(),as shown in Eq.(2).The amount of mature eGFP per cell(P eGFP)is also affected by the degradation rate of mature eGFP(γPeGFP),as shown in Eq.(3).

Absolute protein expression quantity is important for quantitative pathway construction,which has rarely been reported to date.The quantitative measurement of promoter activity that was attained in this study is useful in promoter selection.To verify the usefulness of our method,we selected a three-enzyme metabolic pathway as an example.

The equation system can be solved with Mathematica(Wolfram Research,USA):

例如，在完成《赠汪伦》这一课的教学之后，教师可以利用一张纸遮住“白”“行”“上”“水”“尺”“我”，接着让学生将对应的汉字填进去，谁最先正确地完成谁就获胜。比赛结束后发现，有些汉字具有非常高的出错率，教师基于此可以单独拿出该字，组织学生开展“笔画接力”的游戏，对学生进行分组，让他们以小组为单位来进行比赛。在最后，在教师的引导下对整首古诗进行诵读，让他们的印象得到加深。

where f is a function of the γ,γ,γ,,,n,and t.For

MeGFP IeGFP PeGFP one type of promoter,we regard γMeGFP,γIeGFP and so on as a constant,referred to as A.Next,after analyzing the data of fluorescence from the fluorescence experiments using linear regression,the coefficient(k)between concentration of(C eGFP)and fluorescence intensity(F)can be obtained.

Eq.(12)become the following:

We were able to successfully obtain the purified eGFP.The gel filtration result showed a single peak in the sample(Fig.2(a)).Samples analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)revealed an about 30 kDa induction band(Fig.2(b)).The size of this band was consistent with the predicted mass of the recombinant enzyme.

N colonies were picked.We measured the fluorescence intensity and cell density M times for each colony and then measured the fluorescence intensity and OD Q times for the control(LB medium).The equation can be written as follows:

3.Results

3.1.eGFP purification results

where C meGFP is the concentration of 1 mol eGFP,C cell is the concentration of cells,andεis a constant with a value of 8×108[12].Thus,the promoter activity A can be written as follows:

(3)对于合并糖尿病且服用二甲双胍等双胍类降糖药的受检者,必须在其停药后的48h才安排进行CT检查,且检查后48h才开始服药。

3.2.eGFP stability test

We analyzed by linear regression the results of five independent experiments in which the eGFP was freshly purified.Our findings indicated a strong linear correlation between the fluorescence intensity and the absolute protein concentration(C)measured by BCA assays in our experiment range(r2=0.9733,Fig.3).The results show that the experiment possesses excellent repeatability.

南海争端由来已久，争端所涉及的各方利益错综复杂。几十年来，南海争端的起起伏伏表明，南海各方达成全面和永久性的解决非常困难，非一朝一夕可以完成。然而，南海的和平稳定与繁荣发展事关沿岸数亿人的生产生活，因此在全面和永久解决争议之前，各方积极推进南海海洋合作，是维护海洋和平、增进地区共同福祉的有效途径，也有利于培育各方合作互信的氛围，为实现争端的最终解决做好必要铺垫。

Fig.2.(a)Gel filtration purification of eGFP;(b)SDS-PAGE result of the purified eGFP(31.5 kDa)in Lane 1.The protein was obtained from E.coli BL21(DE3)transformed with pET28a-eGFP.

We measured the fluorescence intensity of each batch of eGFP immediately after purification in order to exclude the impact of fluorescence quenching.The eGFP sample was placed in a black 96-well plate fully covered with aluminum foil before measuring,and was then measured by means of a SparkTM 10 M(Tecan Group Ltd.,Switzerland)provided by the West China School of Pharmacy at Sichuan University.The fluorescence intensity was measured with an excitation wavelength of 450 nm and an emission wavelength of 490 nm.The absolute protein concentration of the purified eGFP was measured using BCA Protein Assay Kit(Sangon Biotech(Shanghai)Co.,Ltd.,China).The experiment was repeated five times to test its repeatability.

3.3.Data analysis

After measuring the OD600 and fluorescence intensity for the measurement device using the method described above,the data were collected and the results were calculated.The original measurement results are documented in the Appendix A(Tables S2 and S3).The absolute protein expression quantity and relative activity of the promoters are shown in Table 2 and Fig.5.

Fig.3.The fluorescence intensity and absolute protein concentration have a linear correlation for the eGFP measured at 450/490 nm.This figure includes data from five independent experiments,which exhibit excellent linear correlation,thus indicating that the experiment possesses excellent repeatability.

Fig.4.The fluorescence intensity and absolute protein concentration have a linear correlation for the eGFP measured at 450/490 nm.This figure includes all the data from the continuous detection experiment,which shows a strong linear correlation.

Table 2 Absolute protein expression quantity and promoter relative activity of the measured promoters.

a The J23100 result was set at 1 for relative activity comparison.

?

Fig.5.Absolute protein expression quantity of the measured promoters.

It is notable that the time-invariance character of the promoter can be demonstrated by the modeling data.According to Table 2,Eq.(4),and the experimental results,the rate of protein production can be determined,as shown in Fig.6.The proportion of accumulated protein expressed by different promoters over different times is the same.Fig.6(b-d)show the protein expression proportion based on the experimental result at a given time.

经过一年多的学习，社团里的大部分社员都能完整地刻出一方印章，姜佳同学还获得过一等奖。社团里的新成员都很羡慕哥哥姐姐能代表学校参加区里的比赛，纷纷铆足了劲儿练习，连放暑假了还要求在学校里继续学习篆刻。

4.Discussion

如果“中国没有向美国明确提出南海是中国的核心利益”观点成立，则“中国能不能捍卫在南海的核心利益？”无需回答(尽管研究结论是明确可信的)，也就不会存在“南海核心利益说”所引起的南海紧张气氛。但实际情况却是，尽管“中国没有向美国明确提出南海是中国的核心利益”观点成立，“南海核心利益说”还是不胫而走，深刻影响南海局势走向。必须明确的是：“南海核心利益说”的始作俑者和推动者是美国媒体、战略界以及政界，其根本目的显然是为美国的南海利益服务。

We set ［ E t］as the total concentration of the enzyme, ［E］as enzyme concentration, ［S］as the substrate concentration, ［ES］as the concentration of the enzyme-substrate complex, ［P］as the product concentration,k1 as the equilibrium constant of the first positive reaction,k2 as the equilibrium constant of the first reverse reaction,and k3 as the equilibrium constant of the second positive reaction[13].

The chemical equation can be written as follows:

Assuming that each part of the reaction has reached equilibrium,the production and degradation rates of ［ ES］are equal.

Fig.6.(a)The modeling result of the time-invariance character of the promoters;(b)the proportion of protein expressed by the promoters after a quarter of the process is complete;(c)the proportion of protein expressed by the promoters after half of the process is complete;(d)the proportion of protein expressed by the promoters at the end of the process.The height of each bar in parts(b-d)represents the proportion of the protein expressed by each promoter to the protein expressed by J23100.

Rearranging this equation gives the following:

Assuming that

Finally,we performed a series of experiments to obtain the relationship between the fluorescence and the concentration,and thus created a scale for the promoters.The molarity of the eGFP(M reCFP)can be written as follows,and it was found to be 31 534.35 Da:

Note that the reaction of［ P］and ［ E］was ignored,even though ［ P］will in fluence the equilibrium,which will affect the production rate.Therefore,as time passes,Eq.(16)may become less accurate.Nevertheless,generally speaking,it is able to describe this process.

Assuming the pathway through which compound A becomes C includes two reactions with two enzymes required,P1 and P2:

法律移植的新语境，就是要关注法律移植后的本土化效果，再进一步来说就是，“排除合理怀疑”的适用要符合我国的司法环境。从中美两国的“异质性”引申出来很多需要解决的问题，才能更好的完善我国的“排除合理怀疑”制度。

Our measurement process followed the routine of the iGEM InterLab study to a large extent[9].All the measurement devices were transformed into E.coli BL21(DE3).Transformed cells were grown overnight at 37 °C and 220 r·min-1,in 5 mL LB medium with 25 μg·mL-1 chloramphenicol.At least three colonies per device were picked on the plate for measurement.The cultures were diluted to a target OD600 of 0.02 in 12 mL LB medium with 25 μg·mL-1 chloramphenicol in a 50 mL falcon tube,and incubated at 37 °C and 220 r·min-1.Three replicates,with 500 μL of LB medium each,were sampled from each tube for measurement after being incubated overnight(16-18 h).Cell density(OD)was measured at a wavelength of 600 nm,and the fluorescence intensity of the bacteria was measured with an excitation wavelength of 450 nm and an emission wavelength of 490 nm(SparkTM 10 M,Tecan Group Ltd.,Switzerland).

Without considering spontaneous degradation,the net reaction rate of one compound(A,B,or C)is equal to its generation rate minus its consumption rate,assuming that the proportions of each reaction are 1:1.

Whether it is a first-order reaction or a zero-order reaction,the rate is only determined by the concentration of enzyme,when the type of enzyme and the initial concentration of the substrate are given(as shown in Eq.(16)).It is apparent that the higher the concentration of enzyme is,the faster the reaction will be.If there is a major difference between the enzyme's maximum rate,V max=k cat［E t］,of two reactions,then the product of one of the reactions cannot be produced,which limits the metabolic rate.Therefore,there is a global best solution to optimize the reactions for multi-step enzyme catalysis(Fig.7).

Fig.7.The concentration variation of substrates.The V max ratio of the three reactions is 1:5:29.

Fig.8.The concentration variation of the substrates.The V max ratio of the three reactions is 1:2:2.

However,if V max is adjusted,the product will not accumulate(Fig.8).Therefore,appropriate promoters must be chosen in order to make V max of each of the three reactions equal.The method described in this paper is an appropriate method of doing so.Given the promoter activity obtained earlier,it is possible to accurately calculate the concentration of protein(C eGFP),which is equal to ［ E］.

烟气再循环技术是近年来的热点问题，康恒、荏原、西格斯等焚烧炉供应商均针对各自炉型进行了相关设计。目前该技术已在国内部分垃圾焚烧发电厂进行应用。以国内2个垃圾焚烧发电厂采用的烟气再循环技术为例，介绍2种不同的工艺方案。

The modeling results indicate that［ E］plays an important role in the metabolic pathway construction.Thus,we have introduced a novel and quick method for absolute protein expression quantitative measurement that will be useful in future applications.Furthermore,the promoter data and gene structure provided by our method can be used directly for metabolic pathway construction in the future.

In addition,we have provided a straightforward way to standardize the strength of different promoters,since the evaluation standard based on the absolute protein quantity can be used directly for all promoters.We have introduced this standardized method and the RiboJ design in order to encourage researchers to characterize promoters using universally comparable parameters(e.g.,absolute protein quantity),and to employ the insulator RiboJ to promote the repeatability and transplantability of gene circuit design in different research studies.Furthermore,the ranking of the relative strength of promoters that was performed in this study differs from the ranking that is based on simple RFP fluorescence intensity measurement,as documented in the Registry of Standard Biological Parts[14].The difference may be caused by the RiboJ design in our research,which can significantly in fluence the expression quantity[7,15].Thus,our method provides greater accuracy in measurement results and gene structure,which will assist pathway construction in the future.

5.Conclusion

In this study,we introduced a novel method to measure the absolute protein expression quantity of a specific promoter.We then utilized our method to successfully measure nine constitutive promoters in the Anderson promoter family,and achieved results that differ from the fluorescent intensity measurement results reported in the literature.Our method provides data with greater accuracy,and offers a straightforward way to standardize the strength of different promoters.

Compliance with ethics guidelines

Hongbin Yu,Zheng Wang,Hanyue Xu,Jiusi Guo,Qingge Ma,Xiangxu Mu,and Yunzi Luo declare that they have no conflict of interest or financial conflicts to disclose.

Acknowledgements

This work was supported by Fundamental Research Funds for Central Universities to Yunzi Luo,the National Natural Science Foundation of China(81502966)and the China Innovation and Entrepreneurship Training Program for Undergraduates(201710611744).We are thankful for the support from our principal investigator(PI)and instructors:Prof.Yunzi Luo,our PI,who provided the lab and supervision necessary for us to finish this project,and Prof.Dan Su,who also provided a lab for us.We thank Huayi Liu and Liping Wang from Yunzi Luo's lab,and Yiping Chen from Dan Su's lab of the State Key Lab of Biotherapy for their advice.We also thank other team members from West China Medical Center of Sichuan University for iGEM 2017,and Wen Guo and Yifan Zhong especially.We thank the West China School of Medicine/West China Hospital and the State Key Lab of Biotherapy for their support.We thank the iGEM foundation for providing the opportunity for us to take part in the worldwide biology competition to start our research.

Appendix A.Supplementary data

所谓的定向钻探，就是在同一个位置多个方位都存在有斜孔，与此同时在斜孔内可以进行定向钻探技术的使用。这一项技术主要应用于深度地质的相关勘查工作，并且主要是在这三个生态环境中：首先是地势较高并且比较陡峭，需要先进行大规模修路工程的地域；其次是在地质的勘查过程中，钻探的深度大于5000米；最后是钻孔直径为65mm，且地质中的岩石中心直径为43mm，可以通过一个钻机场地完成多个方向的钻探工作。所以说，此项技术的应用不仅大幅减少了勘探工程在地表自然环境方面的占地面积，还具有十分显著的绿色地质勘查效果。

Supplementary data to this article can be found online at https://doi.org/10.1016/j.eng.2018.09.012.

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