Quiz 3 - Pt. 1 How Cellular Information is Altered Flashcards
We can alter cells by using ____ or genetic engineering. ____ is subjecting the cells to stress causing changes in the genetic make-up.
mutation
________ is the purposeful transfer of DNA from one type of organism to another.
Genetic Engineering`
mistakes in the genetic code (can arise from replication and/or damage)
Mutations
organism with a genetic mutation
Mutant
the organism without the genetic organism
Wild type
genetic construction of an organism
Genotype
characteristics expressed by an organisms.
Phenotype
usually refers to transcription+translation+posttranslation processing
Expression
EXAMPLE:
- Strain A has the tol operon for toluene degradation, and is in a reactor growing on glucose.
- Strain B has the tol operon for toluene degradation, and is in a reactor growing on toluene.
- These strains have the same _____, but ________
genotype
different phenotypes
base change may or may not result in an amino acid change
Consequences
If the amino acid is the same as before the mutation there is ________.
no consequence
If the amino acid is different, but not in the region of the ______, there may be no consequences
active site
If the mutation is in the active site, there may be some _______________
enzyme activity consequence
If the mutation changes the amino acid to a stop codon, the resulting protein will be _______ and probably ________.
truncated (shortened) and probably not active
confers upon the mutant an advantage for growth, survival or detection under a set of environmental conditions that the wild type does not have.
Selectable mutation
Examples of Selectable Mutation or Selection
- Antibiotic resistance
- Ability to grow on toluene
- Inability to produce lysine
- Ability to produce bioluminescence
- Ability to produce more of an enzyme
- Inability to grow at higher temperatures
Natural Mutation Rates
* 10^-3 - 10^-9 =
* 10^-6 =
mutations per cell conversion
1 mutation/1,000,000 divisions
Increase Mutation Rates
____ : chemicals, radiation
_________ (i.e. lots of divisions)
Mutagens
Lots of growth
Why do we want to increase mutations?
- We want a cell to develop ____________ that are _______ for us.
- For example, ________ inhibition of lysine to increase lysine ______
- specific characteristics, advantages
- removing feed back
- production
uptake of free DNA by a cell. The cell membrane has to be permeable to DNA.
Transformation
DNA is carried into the call in a phage.
Transduction
Cell to cell transfer of DNA. Also called mating.
Conjugation
Natural Gene Transfer/Rearrangement:
Once the DNA is inside the cell it can remain ______ from the chromosome in self-replicating _________ or _____ into the chromosome.
To _____, the DNA must be complementary to the _________________ on the ends.
separate
self replicating plasmid
Integrate
integrate
chromosomal DNA
Using _________ engineers and microbiologists were able to increase penicillin from 0.001 g/L to 50 g/L
mutation and selection
Genetic Engineering:
Using _____ to purposefully manipulate DNA. The DNA is ______ outside of the cell, and then sent into the cell
natural mechanisms
manipulated
Genetic Engineering Tools
Restriction enzymes
Gel electrophoresis (Southern Blot)
Polymerase Chain Reaction (PCR)
Plasmid
enzymes that cut DNA at specific sequences. Different enzymes will cut at different sequences.
Restriction enzymes
A method to detect what sizes of DNA a sample contains.
Gel electrophoresis (Southern Blot)
A process used to make many copies of a piece of DNA.
Polymerase chain reaction (PCR)
self replicating, circular piece of DNA that can survive in a cell.
Plasmid
DNA is denatured by ______
Renaturation on ______
heating
cooling
The ____ cuts both DNA strands at the same site.
DNA fragments join at ______
And Formed a ________
enzyme
sticky ends
Recombinant DNA
Organism from which derived:
- Anabaena Variabilis
- Bacillus amyloliquefaciens
- Bacillus globigii
- Escherichia coli RY 13
- Escherichia coli R245
- Haemophilus aegyptius
- Haemophilus haemolyticus
- Haemophilus inflenzae RD
- Haemophilus parainflanzae
What are the Enzyme called?
Enzyme:
1. Ava I
2. Bam HI
3. Bgl II
4. ECO RI
5. ECO RII
6. Hae III
7. Hha I
8. Hind III
9. Hpa I
Organism from which derived:
- Klebsiella pneumoniae
- Moraxella bovis
- Moraxella bovis
- Providencia stuartii
- Serratia marcescens
- Streptomyces albus G
- Thermophilus aquaticus
- Xanthamonas malvecearum
What are the Enzyme called?
Enzyme:
- Kpn I
- Mbo I
- Pst I
- Sma I
- SstI
- Sal I
- Taq I
- Xma I
A restriction enzyme with _________________________ recognition sequence cuts long DNA more frequently and produce smaller _____________than a restriction enzymes with a six nucleotide recognition sequence.
- restriction enzyme with a four nucleotide
- DNA fragments
Any given ____________ long recognition site occurs in DNA, on average, at a distance of _____ (4^4) nucleotides. Any given _________________ long sequence occurs, on average, at a distance of _____ (4^6) nucleotides. Any given ______ long sequence occurs, on average, at a distance of _____ (4^8) nucleotides
- four nucleotide long recognition site
- 256
- six nucleotide long sequence
- 4096
- eight nucleotide long sequence
- 65,536
_________________ allows scientists to extract and analyze bits of microbial DNA from samples, meaning they don’t need to find and grow whole cells.
Polymerase Chain Reaction (PCR)
PCR is an essential element in DNA ________ and in the sequencing of ________ and the entire ____. Basically, it’s like a technique to photocopy pieces of DNA. In a matter of a few hours, a single DNA sequence can be amplified to ________ of copies. PCR lets scientists work with samples containing even ______ starting amounts of DNA.
fingerprinting
genes and entire genomes
amplified to millions
very small
PRC:
The technique makes use of the DNA repair _______. This enzyme, present in all living things, fixes breaks or mismatched nucleotides in the ______. These breaks or mismatches could cause genes to ____ if left unfixed.
enzyme polymerase
double- stranded DNA helix
malfunction
Not all _______ are created equal, however. Many fall apart in _____.
PCR was developed in ____ following the discovery of an unusual heat-loving bacterium called ________ in a hot spring in Yellowstone National Park.
This bacterium’ s polymerase, dubbed Taq, does its job of ______ and __ nucleotides even in the high heat generated by the successive “____” cycles required during PCR. Taq made PCR possible.
polymerases
high heat
1985
Thermus aquaticus
matching and attaching
“photocopying”
_____ uses the intact half of the DNA molecule as a template and attaches the right nucleotides, which ____ constantly in the cell, to the complementary ____________ at the site of the break.
(DNA consists of two strands of nucleotide bases, which are represented as ______. In the laws of DNA base-pairing, __ joins with __ and __ with __.)
Polymerase
circulate
nucleotide
A, G, C, and T
A joins with T and G with C
Inserting a DNA sample into a Plasmid:
DNA is spliced by ________ pairing and sealed with ______
complementary base
DNA ligase
Are small circles of DNA found in bacterial cells, separate from the bacterial chromosome
Plasmids
cut across the two strands leaving loose ends to which cDNA can be attached
Restriction enzyme
The ____ enters the bacterial cell and reproduces itself. When the bacterials cell divides, the ______ are shared cut between the ______ and the it continue to reproduce.
plasmids
plasmids
two daughter cells
Two type of Bioreactors
Batch and Chemostat (CSTR)
_____: changing conditions - transient (S, X, growth rate), high initial substrate, different phases of growth.
- more common
Batch
____: steady-state, constant low concentration of substrate, constant growth rate that can be set by setting the dilution rate (i.e. the feed flow rate)
- more efficient
Chemostat
Choice of continuous vs.
batch production
- Productivity
- Flexibility
- Control
- Genetic stability
- Operability
- Economics
- Regulatory
Reactor Choices pt.1
- _________: rate of product per time
per volume. Chemostat better for growth associated products. Wasted time in batch process. - _________: ability to make more than one product with the same reactor.
Batch better. - _________: maintaining the same
conditions for all of the product produced. In theory, chemostat better, steady state. In reality????
Productivity
Flexibility
Control
Reactor Choices pt.2
* _________: maintaining the organism with the desired characteristics. Chemostat selects for fast growing mutants that may not have the desired characteristics.
- _____: maintaining a sterile system. Batch better.
- _____: validating the process.
Initially, many process batch, too expensive to re validate after clinical trials.
Genetic stability
Operability
Regulatory
