DNA Synthesis
Vector Selection
Molecular Biology
Oligo Synthesis
RNA Synthesis
Variant Libraries
CRISPR Libraries
Oligo Pools
Virus Packaging
Gene Editing
Protein Expression
Antibody Services
Peptide Services
DNA Data Storage
Standard Oligo
Standard CRISPR Libraries
Standard CRISPR Plasmid
ProXpress
Protein Products
Expression Regulatory Vectors
Expression regulatory vectors usually contain the necessary cis-regulatory elements such as promoters, enhancers, silencers, and one or more cloning sites for inserting the target gene. By designing these regulatory elements, researchers can accurately control the expression level of the target gene at a specific time, in a specific tissue, or under specific conditions.
Main Functions and Applications:
1. Basic Scientific Research: By constructing expression regulation vectors, the expression of the target gene can be accurately controlled at a specific time or in a specific cell type to further study the function of the gene in organisms.
2. Metabolic Engineering: By precisely controlling the expression level of key enzymes, the yield of the target product is increased or the proportion of its metabolites is changed.
3. Pharmaceutical Research and Development: Gene therapy aims to correct or compensate for the function of defective genes by regulating the expression of therapeutic genes, thereby treating diseases at the genetic level.
4. Crop Improvement: By increasing the expression level of drought resistance, disease resistance, or insect resistance genes, the stress resistance of crops is enhanced.
Based on the mode of gene expression regulation, CRISPR-based vectors are primarily classified into CRISPRi (interference) vectors and CRISPRa (activation) vectors. Details are as follows:
|
Vector type |
Characteristics |
KeyComponents |
WorkingPrinciple |
|---|---|---|---|
|
CRISPRiVector |
1. The CRISPR-Cas system is used to suppress or silence the expression of specific genes by preventing the initiation of gene transcription.
2. CRISPRi-mediated knockdown is inducible and reversible. |
dCas9,sgRNA,KRAB |
It mainly depends on the complex of dCas9 (Cas9 without cleavage activity) and sgRNA (single-stranded guide RNA). When this complex is directed to the transcription start site (TSS) of the target gene, dCas9 physically blocks the passage of RNA polymerase, leading to gene silencing. At the same time, dCas9 can fuse a gene inhibition domain, such as the KRAB domain, to further improve the efficiency of transcriptional inhibition. This domain can prevent the binding of transcription factors to DNA, thereby inhibiting gene expression. |
|
CRISPRa Vector |
Gene expression is activated by the CRISPR-Cas9 system. |
dCas9, sgRNA, transcriptional activation domain (e.g. VP64 or p65AD) |
The dCas9 is fused with the transcriptional activation domain (such as VP64 or p65AD) to form a Cas9-activation domain fusion protein with transcriptional activation function. When this fusion protein binds to sgRNA, it can be directed to the promoter or enhancer region of the target gene, and promotes the recruitment of RNA polymerase and the formation of transcription initiation complex through the role of transcriptional activation domain, thereby activating the target gene. |
In Vitro Transcription Vectors
In Vitro Transcription (IVT) vectors,alsoknown as cell-free transcription vectors, is an RNA synthesis process that uses purified DNA as a template in a test tube or other container under laboratory conditions. It is mainly used for the production and research of RNA molecules.
Main Functions and Applications:
1. mRNA Vaccines and Immunotherapy: mRNAs encoding specific antigens can be prepared by in vitro transcription techniques. These mRNAs can be taken up by cells in the body and translated into proteins, thereby inducing an immune response in the body. This technology has been successfully applied to the development of vaccines for a variety of infectious diseases and tumors.
2. Gene Function Research: In vitro transcription is an important experimental tool in the field of basic research and teaching of molecular biology and biotechnology. In vitro transcription experiments can help students and researchers better understand the synthesis, processing, and functional mechanisms of RNA.
3. Biosynthesis and Metabolic Engineering: For the production of RNA molecules with specific functions, such as RNA enzymes, RNA aptamers, etc., these RNAs can be used to produce specific bioactive substances or regulate metabolic pathways.
4. Diagnosis and Detection: IVT vectors can be used to produce RNA molecules labeled with radioisotopes or other probes. By detecting the expression level or structural changes of specific RNA molecules, the pathogenesis and progression of the disease can be assessed.
According to the different in vitro transcription products, they are mainly divided into RNA in vitro transcription vectors and mRNA in vitro transcription vectors. Details are as follows :
|
Vector Type |
NecessaryComponents |
IVT Production |
|---|---|---|
|
RNA In Vitro TranscriptionVectors |
T7 promoter |
RNA |
|
mRNA In Vitro TranscriptionVectors |
T7 Promoter, Kozark Sequence, 5'UTR, 3'UTR, polyA. |
mRNA |
Advantages:
1. Efficient and Fast: Many RNA/mRNA molecules can be synthesized in a short time to meet the experimental needs.
2. High Purity: RNA/mRNA molecules synthesized by the in vitro transcription system are of high purity and do not require complex separation or purification steps.
3. Flexibility: RNA/mRNA molecules of various lengths and sequences can be synthesized, which is suitable for different experimental requirements.
4. Strong Controllability: The in vitro transcription process, reaction conditions, and time can be easily adjusted according to the experimental requirements.
5. Suitable for Large-Scale Production: Due to the high efficiency and controllability of the in vitro transcription system, it is suitable for large-scale production of RNA molecules - such as the production of mRNA vaccines.
Restrictions:
1. Limited Stability: RNA/mRNA molecules transcribed in vitro may be affected by environmental factors such as temperature and humidity during storage and transportation, resulting in decreased stability.
2. Non-Specific Products May Be Produced: During the process of in vitro transcription, some non-specific RNA products may be produced, affecting the accuracy of the experimental results.
3. Not Applicable to All Types of RNA Synthesis: Although in vitro transcription systems can synthesize many types of RNA molecules, effective synthesis may not be achieved for specific RNA structures or functions.
Promoter Function Detection Vectors
Promoter function detection vectors connect the target promoter sequence to the reporter gene (fluorescent protein, enzyme, etc.) and introduces it into the target cell or tissue. When the promoter is activated, it drives the expression of the reporter gene, and the activity of the promoter is evaluated by observing the expression of the reporter gene.
Key Components:
A promoter insertion site and a reporter gene for promoter-driven expression, as well as necessary regulatory sequences and marker genes.
Features:
Reporter Gene Diversity: Usually contains one or more reporter genes, such as luciferase or green fluorescent protein (GFP), used to measure the activity of the promoter.
Application Advantages:
1. Rapid Screening: It can quickly identify and evaluate the activity of the promoter.
2. Quantitative Analysis: The expression level of the reporter gene can be quantified to facilitate the comparison of the efficiency of different promoters.
3. Diversity Assessment: Suitable for a variety of research applications, including promoter identification, functional analysis, and optimization.
4. Technical Maturity: Experimental design ideas and details such as transcriptional regulatory element activity and binding site prediction using a dual luciferase reporter system are very mature.
Restrictions:
1. Background Noise: Some vectors may have non-specific expression, resulting in background noise.
2. Host Restriction: The activity of the promoter may be affected by the type of host cells.
3. Cost: It may be costly to purchase or construct a highly specific detection carrier.
Protein Interaction Detection Vectors
Protein interaction detection is usually achieved by constructing a fusion protein expression vector, in which the target protein sequence is fused with a reporter gene or a tag sequence, and the two fusion proteins are co-expressed in host cells. When these two fusion proteins interact, they can activate or inhibit the reporter genes that are linked to them, thereby triggering measurable signal changes, such as enhanced fluorescence intensity or color changes. By detecting these signal changes, researchers can assess and quantify protein-protein interactions.
Key Components:
Target gene insertion sites, reporter system elements for reporting protein interactions.
Features:
1. Dual Reporter Gene System: Usually contains two reporter genes to monitor the interaction between proteins.
2. Fusion Tag: The vector design contains a fusion protein tag, such as GST, His, or Flag, to facilitate protein purification and detection.
3. Signal Amplification: Some carriers improve the sensitivity of detection by signal amplification mechanisms.
Advantages:
1. Real-Time Monitoring: The dynamic process of protein interaction can be monitored in real time.
2. Quantitative Analysis: A method for quantitative analysis of protein interaction intensity is provided.
3. High-Throughput Screening: Suitable for large-scale screening experiments and helps to quickly identify interacting proteins.
4. Widely Used: Can be used for drug screening, disease mechanism research, and signal transduction pathway analysis.
Restrictions:
1. False Positive / False Negative: May be due to non-specific binding or improper experimental conditions lead to wrong results.
2. Limitations: Protein interaction detection vectors usually can only detect the interaction between known proteins and may not be able to detect unknown protein interactions. In addition, some low-affinity or transient protein-protein interactions may not be detectable.
One-stop Solution for Vector Construction |Synbio Technologies
Synbio Technologies has authored a detailed and clearly structured vector guide designed to provide a convenient reference tool for researchers. In the vector guide, we introduce many types of vectors, including but not limited to plasmid vectors, viral vectors, cloning vectors, expression vectors and so on. We provide detailed descriptions of each vector, including its features, advantages, limitations, applications and other key information.
Synbio Technologies offers comprehensive vector libraries. With 200+ standard vectors, we provide customers with a wide selection of vectors. We expertise includes the design, construction and optimization of plasmid vectors for various applications. The vectors are efficient, stable and can carry a large number of foreign DNA inserts. Whether you are in research, drug discovery or industrial biotechnology, we has the vectors and expertise you need to succeed.
Meanwhile, Real-time vector editor allows users to flexibly and conveniently remodel vector mapping sequences online according to their specific needs. With the intelligent search function, users can quickly and easily filter out the carrier atlas that meets their requirements based on keywords such as carrier number, carrier function, and species.
