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Genes that are present in a cell
the expression of a gene
- deoxyribosenucleic acid
- 4 bases: Adenine-Thymine, Guanine-Cytocine
- 4 bases: Adenine-Uracil, Guanine-Cytocine
% of DNA that codes vs. non-coding
- Coding: 2%
- Non-coding: 98%
- Enzyme divides strands of DNA
- DNA polymerase is used to copy continuously from 5'-3'
- Okazaki fragments fill in from 3'-5'
- DNA being copied to mRNA
- Occurs in nucleus
- requires RNA polymerase
Ribosome reads mRNA and creates proteins from amino acids coded in mRNA
Coding sequences only in eukaryotic DNA
Non-coding sequences in eukaryotic DNA that are cut out.
structure of a gene
an operon that can be turned on and off
- cells prefer glucose as a fuel
- When glucose is present: cAMP levels are low
- When glucose is absent: cAMP levels are high
- any change in DNA sequence
- can be good, harmful, neutral or deadly
mutation codes for same amino acid, so organism is unaffected
- codes for different amino acid
- could be good or bad
- adds a premature stop codon
- usually deadly
- addition or deletion of a base, changing sequence after
- usually deadly
Spontaneous Mutation rate
1 in 1,000,000 (10-6)
- 1) Spontaneous mutation
- 2) Mutagens
- 3) Radiation
- 4) Certain Viruses
- Increases mutation rate by 10-1000x
- benzopyrene (burnt stuff)
- aflatoxin (PB in other contries)
Effect of UV on DNA
- sunburn causes thymine dimers.
- Two T's attach to each other instead of A's
- Excision repair fixes it
Example of Mutation Virus
Positive (Direct) Selection
- ex. testing antiobiotic resistance
- Putting bacteria on antibiotic plate. If any growth they are antibiotic resistant.
- Positive selection entails the growing the culture on a medium that will alow for the growth of only the mutant colonies
.If, for example, we want to find mutants that resistant to penicillin, we grow the culture on a medium that contains pencillin. Only those colonies that are resistant to penicillin will grow and we ca identify them directly
Negative (In-direct) Selection
- Negative selection is used to identify mutants that have lost the ability to perform a certain function that their parents had.
- -Auxotrophic mutants, for example, are bacteria that have lost the ability to synthesize an essential nutrient.
- The replica-plating technique is used to identify mutants by negative selection.
- -the replica-plating technique can be used, for example, to identify mutants that have lost the ability to synthesize the amino acid histidine. Therefore, mutants are His- and require histidine in order to survive.
- `Inoculate a histidine enriched medium with bateria. Incubate so that cells can form colonies. This is the master plate.
- `Press a sterile velvet surface into the colonies of the master plate. Some cells from each of the colonies adhere to the velvet.
- `Prepare two mediums, one with histidine, the other without histidine.
- `Transfer cells from the velvet to each plate.
- `Compare growth on the two plates after incubation. Colonies that grow on the histindine enriched medium but not on the medium lacking histidine are His- mutants.
using sterile velvet to move organisms to different plates
- Good at testing out potential carcinogens.
- Reverses gene mutations
Uptake of naked DNA
F+ bacterial cell transfering DNA to F- bacterial cell via pili
- Virus takes over cell and makes new viruses inside infected cell
- Sometimes they accidently pick up host DNA and bring it to a new host
- Jumping genes.
- Segment of DNA that can cut itself out and move to another chromosome
- Indian corn
Chemicals some bacteria produce to harm other bacteria
- use of microbes for a desired outcome
- ancient-beer, cheese
Recombinant DNA Technology
- moving genes from one organism to another to get the gene expressed
- new since 1970s
- first time: putting firefly genes in tobacco to make it glow
Site Directed Mutagenesis
Trying to mutate cells to create a better geneotype
- cut DNA at specific sequences
- Can be blunt cut or staggered cut
a circular vector piece of DNA
viruses that infect bacteria
Polymerase Chain Reaction
- amplifies DNA from one strand to billions in 3 hours
- requires heat
- DNA is split, primers are added and DNA polymerase copies DNA
Blue White Screening
Proves that you are successful in getting DNA into e. Coli
No growth Result of B/W screening
Bacteria did not take up vector
Vector got ito bacteria, but not DNA
- Both Vector and DNA entered cell
uptake of DNA
put DNA in test tube and give it an electric shock
- Enzymatically remove cell walls from 2 cells and put it in dense glycerine solution
- causes cells to fuse together into 1 cell
gun that coats DNA in gold bullets and shoots into cells
- Injectiing DNA into cell
- Holds pieces of DNA and cells
- DNA is cut up, put in vectors, put in e. coli and deep frozen
- Turns RNA into cDNA
- HIV virus
Examples of Therpeutic Genetic Engineering
- Subunit vaccines
- gene therapy
- DNA vaccines
- Gene scilencing
Examples of Scientific Genetic Engineering
- reverse genetics
- human genome project
- human microbiome project
- DNA fingerprinting/forensics
Examples of Agricultural Genetic Engineering
- more nutritiotous animals and plants
- dourght resistance
- salt resisntace
- pseudomonas syringae-prevents frost damage
- bacillus thuriengenesis-kills mosquito larvae