MindMap Gallery Genetic Engineering
This is a mind map about genetic engineering. According to people's wishes, through transgenic and other technologies, we can give organisms new genetic characteristics and create new types of organisms and biological products that better meet people's needs.
Edited at 2024-03-25 10:55:46One Hundred Years of Solitude is the masterpiece of Gabriel Garcia Marquez. Reading this book begins with making sense of the characters' relationships, which are centered on the Buendía family and tells the story of the family's prosperity and decline, internal relationships and political struggles, self-mixing and rebirth over the course of a hundred years.
One Hundred Years of Solitude is the masterpiece of Gabriel Garcia Marquez. Reading this book begins with making sense of the characters' relationships, which are centered on the Buendía family and tells the story of the family's prosperity and decline, internal relationships and political struggles, self-mixing and rebirth over the course of a hundred years.
Project management is the process of applying specialized knowledge, skills, tools, and methods to project activities so that the project can achieve or exceed the set needs and expectations within the constraints of limited resources. This diagram provides a comprehensive overview of the 8 components of the project management process and can be used as a generic template for direct application.
One Hundred Years of Solitude is the masterpiece of Gabriel Garcia Marquez. Reading this book begins with making sense of the characters' relationships, which are centered on the Buendía family and tells the story of the family's prosperity and decline, internal relationships and political struggles, self-mixing and rebirth over the course of a hundred years.
One Hundred Years of Solitude is the masterpiece of Gabriel Garcia Marquez. Reading this book begins with making sense of the characters' relationships, which are centered on the Buendía family and tells the story of the family's prosperity and decline, internal relationships and political struggles, self-mixing and rebirth over the course of a hundred years.
Project management is the process of applying specialized knowledge, skills, tools, and methods to project activities so that the project can achieve or exceed the set needs and expectations within the constraints of limited resources. This diagram provides a comprehensive overview of the 8 components of the project management process and can be used as a generic template for direct application.
Genetic Engineering
basic
definition
According to people's wishes, through technologies such as genetic modification, new genetic characteristics are given to organisms to create new types of organisms and biological products that better meet people's needs.
principle
Theoretical basis of gene splicing
DNA is genetic material
The building blocks of DNA are dNTPs
The spatial structure of DNA is a regular double helix structure
Theoretical basis for expression of foreign genes in recipients
Genes are independent units of inheritance
The transmission of genetic information follows the central dogma
share a genetic code
Basic tools of recombinant DNA technology
restriction endonuclease "Molecular scalpel"
primary source
Isolated and purified from prokaryotes
effect
Recognizes specific nucleotide sequences in double-stranded DNA molecules and breaks the phosphodiester bonds at specific locations in each strand
eg: EcoR I can recognize G↓AATTC, Sma I can recognize CCC↓GGG
cutting form
sticky ends
flat end
DNA ligase "Molecular suture needle"
effect
Join double-stranded DNA fragments to restore the phosphodiester bond between two nucleotides cut by restriction enzymes
Classification
E.coli DNA ligase
source
Isolated from Escherichia coli
Features
Can only join DNA fragments with complementary sticky ends
T4 DNA ligase
source
Isolated from T4 bacteriophage
Features
Can connect both complementary sticky ends and blunt ends, but blunt ends are less efficient
vector for gene entry into recipient cells "Molecular Transporter"
Commonly used carriers
Plasmids, phages, and animal and plant viruses
Plasmid: a naked, simple-structured, circular double-stranded DNA molecule that is independent of eukaryotic cell nuclei or prokaryotic cell nucleoid DNA and has the ability to self-replicate.
Require
① Harmless to receptor cells and does not affect their normal life activities
②Can be replicated and stably stored in recipient cells
③Have marker genes for screening and identification
④Have more than one restriction enzyme cutting site
DNA extraction and identification
Experimental principle
①DNA is not soluble in alcohol, but some proteins can - preliminary separation of DNA and proteins
②DNA can be dissolved in 2mol/L NaCl solution
③DNA appears blue when exposed to diphenylamine reagent
method
Grind
Remove impurities
After filtration, cool/centrifuge and take the supernatant.
extract
After adding 95% alcohol solution, white thread-like objects appear in the solution → roll up/centrifuge with a glass rod, take the precipitate and dry it
identification
Identification using diphenylamine reagent - precipitate/filament appears blue
Basic operations of genetic engineering
Screening and acquisition of target genes
Screen for suitable target genes
definition
Genes used to change the characteristics of recipient cells or obtain expected expression products, etc.
effect
Relevant to biological stress resistance, good quality, production of pharmaceuticals, non-toxic degradation and industrial enzymes
e.g.
Bt insect-resistant protein gene - derived from the Bacillus thuringiensis companion crystal protein produced by Bacillus thuringiensis, which can kill cotton bollworms
filter
Screen suitable target genes from related genes with known structures and clear functions
Use PCR (polymerase chain reaction) to obtain and amplify the target gene
definition
A technology based on the principle of DNA semi-conservative replication, which provides various components and reaction conditions involved in DNA replication in vitro, and replicates the nucleotide sequence of the target gene in large quantities.
Basic conditions
high temperature
Open DNA double strands
DNA parent strand
Provides a template for DNA replication
dNTP
Provide raw materials for synthesizing sub-chains
DNA polymerase
Catalyzes the synthesis of DNA daughter strands
primer
Enables DNA polymerase to ligate deoxynucleotides starting from the 3' end of the primer
ATP
helicase
composition direction
Extend from the 5' end of the daughter strand to the 3' end
reaction process
transsexual
When the temperature is >90°C, double-stranded DNA depolymerizes into single strands
Restoration
When the temperature drops to about 50°C, the two primers combine with the two single-stranded DNA through complementary base pairing.
extend
When the temperature drops to about 72°C, ATCG synthesizes new DNA strands under the action of TaqDNA polymerase according to the principle of complementary base pairing.
Construction of gene expression vectors
Purpose
Let the gene exist stably in the recipient cell and be passed on to the next generation
Enable the target gene to express and function
composition
Promoter
The site where RNA polymerase recognizes and binds to drive gene transcription into mRNA
terminator
Stop the transcription at the desired location
marker gene
Identify whether the recipient cells contain the target gene, thereby screening out the cells containing the target gene
target gene
copy origin
build process
① Use a certain restriction enzyme to cut the vector to create a nick
② Use the same restriction enzyme or a restriction enzyme that can produce the same end to cut the DNA fragment containing the target gene
③ Use DNA ligase to splice the target gene fragment into the nick of the vector to form a recombinant DNA molecule
Basic structure of eukaryotic cell genes
The target gene generated by reverse transcription does not have introns, promoters and terminators, so RNA polymerase in prokaryotic cells cannot recognize the promoter sequence of eukaryotic genes.
tips
Double enzyme digestion can be used to prevent reverse ligation or self-ligation.
Introduce the gene of interest into the recipient cells
Import plant cells
pollen tube passage method
method
Use a microsyringe to directly inject the DNA solution containing the target gene into the ovary
After pollination, the stigma is cut off, and the DNA solution is dripped onto the cut surface of the style so that the target gene can enter the embryo sac through the pollen tube channel.
Agrobacterium transformation method
step
① Insert the target gene into the T-DNA of the Ti plasmid to obtain a recombinant plasmid containing the target gene
②Transfer this recombinant plasmid into Agrobacterium to obtain the recombinant plasmid Agrobacterium
③Introduce Agrobacterium into plant receptor cells
④T-DNA is transferred to the recipient cell and the target gene is integrated into the DNA chromosome of the recipient cell for stable maintenance and expression.
gene gun method
Using the power generated by compressed gas, the expression vector DNA wrapped on the surface of the metal particles is driven into the receptor, allowing the target gene to be integrated and expressed.
Import animal cells
microinjection
Microinjection is performed using a microinjector to inject the target gene into the fertilized egg of the animal, and then transplanted into the fallopian tube or uterus of the female animal, so that it can develop into an animal with new traits.
Introduction of microbial cells
Ca²⁺ treatment
Treat cells with Ca²⁺ to make them competent cells and absorb DNA molecules in the peripheral environment to complete the transformation
Detection and identification of target genes
molecular level
import
PCR technology detects whether the target gene is inserted into the chromosome of the recipient cell
Transcribe
PCR technology detects whether the target gene has been transcribed into mRNA
translate
Antigen-antibody hybridization technology detects whether the target gene translates into a specific protein
individual level
Pest-resistant plants – pests eat – whether pests die
Disease Resistant Plants – Virus Infection – Whether Symptoms Appear
Salt/Herbicide Tolerant Plants – Salt Water/Herbicide Watering – Are They Growing Normally?
Whether the genetic organism from which the target gene product is obtained - extracted and compared with the natural product - is normal
Applications of genetic engineering
Agriculture and animal husbandry
Improve plant quality
Transgenic insect-resistant plants
Transgenic disease-resistant plants
Genetically modified herbicide-resistant plants
Improve plant quality
Improve animal quality
Increase animal growth rate
Improve the quality of livestock products
Medicine & Health
Using genetically modified microorganisms, animals and plants to produce drugs
Using mammals to mass-produce drugs
Breast (room) bioreactor
Realizing the idea of establishing a transplant organ factory
Gene therapy
extracorporeal therapy
Gene defective virus is isolated from the body → transformed and amplified in vitro → transferred into the body (lymphocytes)
In vivo treatment
The vector carrying the normal gene is directly transferred into the patient's tissue cells (lung tissue)
food industry
Industrial amino acids
industrial enzymes
Isolate target genes from gene libraries
Genes in cDNA: small base and short genes