Team:BIT/Biology
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Background
Breast cancer is one of the most common malignant tumors among women. About one million and two hundred thousand women suffer from breast cancer and five hundred thousand women die of breast cancer every year around the world. The morbidity of breast cancer is always the top of the list. However, the cure rate of breast cancer is higher than that of others. In Western Europe, North America and other developed countries, the morbidity of breast cancer is the highest among women who suffer from cancer. In America, per one hundred thousand people, there are 129.9 breast cancer patients. This number is 48.16 in Japan, 43.71 in Hong Kong, 17.09 in China and 13.94 in Korea.
Biomarkers usually refer to indicators that are used to measure physiological state or condition. Biomarkers have many applications in measuring and evaluating physiological processes, pathogenic process or in the process of drug treatment. At present, it has important significance not only in the medical field, but also in the field of molecular epidemiology, molecular toxicology and environmental medicine.
MicroRNAs are a class of small, non-coding RNA molecules, containing about 22 nucleotides. MicroRNAs fold itself in a way similar to RNA interference (RNAi), forming a short hairpin structure. It is the precursor substance which Single-stranded RNA forms by folding itself. Then enzyme DICER cleaves it into miRNA (about 22 nt in length) [17-18]. MicroRNAs mainly have three ways to regulate the expression of gene. The first way is to cleave mRNA of the target gene. miRNA completely complementary combine with target genes and cleave mRNA finally. The mechanism and function is very similar with siRNA. The second way is to inhibit the effect of the translation of target genes through incomplete combination with target genes. In this way translation is suppressed without affecting the stability of the mRNA. The third way Is the combination of the above two methods. When miRNA completely complementary combine with target genes, the target mRNA will be cleaved. When miRNA incompletely complementary combine with target genes, miRNA play a role of regulating the expression of gene. Research shows that the levels of many kinds of microRNA in the sick body are different. Besides, for different diseases, the levels and types of miRNA are different. In other words, miRNAs can be disease markers. Among the vast majority of cancer patients, in their body the levels of miRNAs change compared with the normal level. For example, In the human body suffering from breast cancer, the levels of miR-21、miR-155、miR-244、miR-139 increase, while the levels of miR-497、miR-31 decrease.
Principle
1. Live cell Logic gate genetic components
Bonnet and Siuti, who use recombinant enzymes to build a logic gate which is different from the traditional logic gate routes. They insert phage DNA into the host genome integrase site. The integrase recognizes a pair of distinct sequences, called attP (on phage) and attB (in the host), which are different from each other. After recombination and insertion of the phage, attP and attB are flipped and converted into a pair of sites called attL and attR, which are no longer substrates for the recombinase. Using attP and attB sites, deliberately construct a modified DNA sequence, you can "cheat" recombinase, so that the DNA sequence occurs to an irreversible reversal.
In the figure, the GFP protein is expressed in the "0" state. However, when the recombinase site was specifically inverted, it became "1" and the left RFP protein was expressed
2. RNA-based gene silencing expression
RNA interference and related gene silencing led to changes in the metabolic pathway has completely changed people's understanding of gene regulation. Gene silencing technology has been used as a research tool to control the expression of certain cell genes. In eukaryotes, RNA interference manipulates many cellular functions as another way of gene silencing. Especially in mammals, it can regulate gene expression, analyse signal transduction gene interaction, block the pathogenesis of the disease gene. Zheng Xie[30] et al. designed a transcriptional (post-transcriptional) regulatory gene pathway, which can sense the expression of endogenous microRNAs and cause intracellular reactions only by sensing the level of microRNA expression level. The schematic diagram below designs a sequence that specifically binds to the corresponding microRNA downstream of the gene line and gives a final output signal by sensing the level of microRNA expression.
3. Riboswitch
Riboswitch plays an important role in gene regulation in the prokaryotic system. Riboswitch achieve the "switch" function mainly by changing ribonucleic acid (RNA) conformation, prevent or open the target protein production. Most Riboswitches have only one binding site or aptamer that recognizes a targeting ligand. Aptamers are usually located near the gene expression region. When combined with metabolites, it will change its structure and exercise the function of gene regulation at the level of transcription or translation. Riboswitch can be "turned on" and re-activated for transcription or translation by a regulatory sRNA (here referred to as a small fragment of regulatory RNA, a non-coding RNA that is important for eukaryotic and prokaryotic regulatory action) [31]. F.J. Isaacs et al. in J. J. Collins task group constructed a modular ribosome switch. They first inserted a short nucleotide sequence complementary to the RBS upstream of the DNA, named the cis-suppression sequence, located between the 5'-UTR and RBS sequences downstream of the promoter. The insertion of the cis sequence does not alter the coding sequence of the target gene, nor does it affect the rate of transcription. The cis sequence consists of two parts: a hairpin sequence consisting of 19 nucleotide residues complementary to the RBS and a loop domain consisting of 6 nucleotide residues. The 5'-UTR of the crRNA (cis-repressed MRNA) produced by transcription is capable of folding and masking RBS, thereby preventing the translation of functional proteins [32]. The principle is as follows.
Design
In the system we designed, we introduced three unique and innovative parts including gene silence part based on microRNA, Lock & Key part and double integrases part. We use them to achieve joint detection of breast cancer-related markers microRNA-21 and microRNA-155. The system principle as follows:
When microRNA-155 and microRNA-21 are absent or low expression, the tetR and LacI proteins are expressed under the promoter of the constitutive promoter, thereby inhibiting the initiation of the downstream of the ptet promoter and the plac promoter, so that the expression of FimE and BxBl recombinase is not expressed or low expression. At this time, downstream integrase site does not occur specific inversion.
When microRNA-155 and microRNA-21 were present, the specific binding of microRNA and mRNA (transcription of binding sites) prevented the expression of tetR and LacI proteins. Thus, the inhibition of ptet promoter and plac promoter was released and recombinase was expressed, recognition site-specific flipped, site-controlled terminator was flipped, downstream gene expression was opened. As a result, fluorescent protein was expressed by reporter gene.
In addition, we add ribosome switches before the expression of FimE and BxB1, which aims to reduce the background noise expression of FimE and BxB1 recombinase and enhance the stability of the system.
The advantages of the system are as followed: First of all, the features of gene silence make the detection of microRNA possible and effective in the engineering bacteria. Secondly, employing the Lock & Key part in our system reduce the background noise in order to achieve the lower limit of detection. Ultimately, the joint determination of microRNA-21 and microRNA-155 is feasible by the design of double integrases part. All in all, the system realizes the highly sensitive simultaneous determination of microRNA-21 and microRNA-155, which are related to the breast cancer.
Contact Us
Beijing Institute of Technology,
No. 5 South Zhong Guan Cun Street,
Haidian Beijing 100081, P. R. China
Twitter : @igem_BIT
Sina Weibo : @igem_BIT
Website : http://www.bit.edu.cn
