The components of a qualified plasmid include:
1. The copy start site Ori is the site that controls the start of replication. There is only one origin of replication in prokaryotic DNA molecules. The eukaryotic DNA molecule has multiple replication initiation sites.
2. Antibiotic resistance genes can be easily detected, such as Amp, Kan
3. Multiple cloning site MCS clones carry foreign gene fragments
4. P/E Promoter/Enhancer
5. Terms termination signal
6. Adding poly(A) signal can stabilize the mRNA function and read the plasmid map.
The first step: first look at the location of Ori
Understanding the type of plasmid (prokary/eukaryotic/shuttle plasmid) The so-called shuttle plasmid refers to a type of artificially constructed plasmid vector that has two different origins of replication and selection markers and thus can survive and replicate in two different taxonomic hosts. This concept is not only used for different microbial flora, but also for the construction of eukaryotic expression vectors, such as pBE2 for subtilis, pPIC9K for yeast, mammalian expression vector pMT2, and Ti plasmid for plant cells. These shuttle plasmids can be expanded and expressed not only in E. coli but also in corresponding subtilis, yeast, animal or plant cells. This facilitates molecular biological manipulation and bulk preparation of the plasmid.
Step 2: Look at the filter mark again
If resistance, decide what filter to use.
1. Ampr hydrolyzes the β-lactam ring to relieve the toxicity of ampicillin. Shanghai Chuangsai Technology provides ampicillin/ampicillin, >980 mcg/mg, USP, BR, which can be used for cell culture, product number: C84-1507-5G, price 78 yuan.
2. tetr prevents tetracycline from entering the cell. Shanghai Chuangsai Technology provides tetracycline hydrochloride, ≥900μg/mg, USP32, BR, 64-75-5, commodity number: C84-2004-10G, price 108 yuan.
3. camr produces chloramphenicol hydroxyacetyl derivatives, rendering them toxic.
4. neor(kanr) aminoglycoside phosphotransferase inactivates G418 (naphthomycin derivative)
5. hygr inactivates hygromycin beta. Shanghai Chuangsai Technology provides hygromycin B, >90%, powder, score, 31282-04-9, commodity number: C84-6158-100MG, price 200 yuan.
Step 3: Look at the multiple cloning site (MCS)
It has a single point of multiple restriction enzymes. Facilitate the insertion of foreign genes. If there is an insertion of a foreign gene outside these sites, it will result in the inactivation of a certain marker gene and facilitate screening. Decide whether you can put the target gene and how to place the target gene.
Step 4: Look at the size of the foreign DNA insert
Plasmids generally can only accommodate foreign DNA fragments of less than 10 Kb. In general, the longer the foreign DNA fragment, the more difficult it is to insert, the more unstable it is, the lower the transformation efficiency.
Step 5: Whether to contain expression system components
That is, the promoter-ribosomal binding site-cloning site-transcription termination signal. This is used to distinguish between cloning vectors and expression vectors. Adding some elements related to expression regulation to the cloning vector becomes an expression vector. The choice of the carrier is still based on the purpose of the experiment. Promoter-ribosomal binding site-cloning site-transcription termination signal.
1. Promoter: A DNA sequence that promotes DNA transcription. This DNA region is often upstream of the coding sequence of a gene or operon. It is a site on the DNA molecule that specifically binds to RNApol and initiates transcription, but the promoter itself is not Transcription.
2. Enhancer/silencer: One of the eukaryotic genomes (including the eukaryotic viral genome) has a regulatory sequence that enhances the transcription process of adjacent genes. Its role is independent of the position or orientation of the enhancer. That is, it can play a role upstream or downstream of the regulated gene. Silencer-negative enhancer, negative regulatory sequence.
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