PCR and Bacterial Transformation Test
Calcium Chloride Transformation procedure (4 steps)
1. Bacteria are washed in ice cold calcium chloride, which makes it more permeable 2. Plasmid DNA is added to the cells and the mixture is incubated on ice 3. The cells are heat shocked in a water bath and placed back on ice 4. Non-selective growth media is added to the cells
How to find transformation efficiency?
1. DNA spread=(volume spread x DNA in transformation)/total volume of transformation 2. Transformation efficiency=number of CFUS/DNA spread on plate
What are the three stages of PCR?
1. Denaturation 2. Annealing 3. Extension
What are the three steps to creating a transgenic animal?
1. Gene of choice is manipulated and prepared in lab 2. Transgene is injected into the egg of an animal 3. Egg is implanted into a sureogate
Bacterial Transfection procedure (2 steps)
1. Plasmid DNA is sealed in tiny oil bubbles (lipid vesicle) and incubated in cultured medium 2. The vesicles fuse with the cell membranes and deliver DNA into the cells
How long can PCR strands be?
100s to 1000s of bases long
When was the first use of plasmids? How did they use it?
1978 by Genetech Used the gene that codes for human insulin and expressed it in bacteria
What is the Forward Primer?
1st primer; binds at the beginning of the target region of the DNA
What is the Reverse Primer?
2nd Primer; binds at the end of the target region
What direction does extension read? Write?
3' to 5' 5' to 3'
What is the range of copy number?
5 (low) to 1000 (high)
What can be added into a gene while the gene is in a plasmid?
A new restriction enzyme site
What do plasmids contain?
A promoter and a terminator
What is a promoter?
A sequence of DNA that is located before the protein-coding sequence and provides a landing site for the RNA polymerase so that the gene can be transcribed and the protein and be made
What is a terminator?
A sequence of DNA that tells the RNA polymerase to STOP transcribing the gene so that the mRNA is the correct length
What is PCR?
A simplified version of bacterial DNA replication that copies a specific sequence of DNA (the target sequence) so that it is amplified
What are Multiple Cloning Sites?
A specific area in cloning plasmids that is a string of restriction enzyme recognition sites to make cloning as easy as possible
What are the three steps of genetic engineering?
Amplify plasmids Purify Analyze/quantify
What happens during stage 2 of PCR?
Annealing The primer binds the target sequence by cooling the single stranded DNA to allow hydrogen bonding between the primers and the DNA without allowing the DNA to become a double helix
What are enzymes in natural plasmids resistant to?
Antibiotics
What is transduction?
Bacteria does not need to physically contact one another to transfer genetic information. Instead it is transferred by bacteriophages that infect the bacteria
What is smooth bacteria?
Bacteria encapsulated in a polysaccharide coat (lethal)
What is rough bacteria?
Bacteria not encapsulated in a polysaccharide coat (not lethal)
Plasmids allow a gene to?
Be moved from one organism and expressed in another
Why are different temperature used in PCR?
Because DNA changes between single and double strands depending on temperature
What is the Annealing Temperature?
Between 50-60 deg C, but can vary based on length of sequence or primer
What are nucleotides?
Building blocks for new strands of DNA that provide a power source of the reaction
What is Degenerate PCR?
Can amplify DNA when target sequence information is limited or when the same PCR needs to work with DNA templates from different species
What is a cloning plasmid?
Carry/house genes that have been cloned
What is the structure of plasmids?
Circular
What are the two main types of plasmids?
Cloning plasmids and expression plasmids
What is Master Mix?
Combination of DNA polymerase, dNTPS, primers, and reaction buffer
What is the Lac Operons?
Controls the production of 3 enzymes that are involved in the metabolism of lactose in E. coli
What is the araBAD operon?
Controls the production of enzymes involved in the metabolism of arabinose
What are Amplicons?
Copies produced by PCR
What is DNA replication?
Copies the entire genome of bacteria
What is Fast PCR?
Cute the standard running time of 2.5-4 hr to 0.5-1.5 hr by choosing primers that combine annealing and extension
What recognition site does ori have?
DNA polymerase
What is magnesium chloride essential to?
DNA polymerases
What happens during step 1 of PCR?
Denature Template DNA is heated to 94 deg C which denature the double stranded DNA due to a breakage of hydrogen bonds between base pairs
What is Bacteria Conjugation?
E. coli is shuttle DNA across a bridge that forms between the cells. This is dependent of fertility and only F+ bacteria can transfer DNA
What does transcription of genes require?
Energy
What is an expression plasmid?
Express recombinant proteins aka a protein is to be made from a gene
What happened during stage 3 of PCR?
Extension DNA polymerase extends the primers by reading the complimentary strands and adding matching nucleotides
What are plasmids?
Extrachromosomal circular loops of DNA that can replicate themselves in the cytosol of bacteria
What is F+ and F- Bacteria?
F+: has hairs F-: is hairless
What is it called when bacteria divide?
Fission
What two other fields has PCR transformed?
Forensic analysis and archeology
How has PCR transformed agriculture?
GMOS and genetically modified animals to create drugs
What is a constitutive gene?
Genes that are always expressed
What is a facultative gene?
Genes that are transcribed only when needed
What type of template DNA is used in PCR?
Genomic, plasmid, or complementary
What are B-lactamases?
Groups of enzymes with the bla gene that are able to break down antibiotics with B-lactam rings like penicillin and ampicillin
What does genetic engineering require?
Introduction of new DNA into cells using plasmids and virus
What is the biggest con of PCR?
It is prone to contamination without additional precautions
What does it mean if a bacteria has bla?
It will grow in the presence of antibiotics
Who discovered PCR?
Kary Mullis in 1983 while driving down the highway
What is cloning?
Making exact copies of the DNA sequence of a gene
What is Random Amplification of DNA?
Method used when the genomic sequence is unknown, uses several primers to identify similarities in the sequence to known ones; often used in forensics
How many copies of plasmids can be made when growing them in bacteria?
Millions
Are plasmids more or less stable than linear DNA?
More stable
What is Multiplex PCR?
Multiple target sequences are simultaneously detected
What are operons?
Naturally occurring control units in bacterial chromosomal DNA that consist of one promoter, multiple genes, and a single terminator
How do plasmids occur?
Naturally or engineered to serve specific research goals
How is the expression of facultative genes regulated in bacteria?
Operons
What is the most common technique used to clone plasmids?
PCR
Are plasmids or linear DNA more easier to manipulate?
Plasmids
What is the method of bacterial transformation?
Plasmids are mixed with the bacteria and enter cells. The cells are then plated on selective mediums so only the bacteria that receives the plasmid will groe
What type of bacteria was used to discover transformations?
Pneumonia
What does PCR use to target specific sequences of DNA?
Primers and thermophilic DNA polymerase
What is a mini prep?
Production of plasmids on a small scale
Which cells have operons?
Prokaryotes
What is a PCR buffer?
Provides a suitable environment for the cations involved and maintains a pH that is optimized for the enzyme
What are plasmids the starting point for?
Recombinant protein production in drug development
What does DNA polymerase do?
Replicate the DNA and enable the plasmid to be cloned as the bacteria divides
What type of PCR is used for Covid testing?
Reverse transcription PCR
What bacteria was used to discover transduction?
Salmonella
What are primers?
Short strands of DNA that match and bind each end of the target sequence
What does PCR do?
Specifically replicates targeted strands of the DNA lots of times
What are operons induced by?
Sugar
What are inducers?
Sugars the induce operons
What enzyme is typically used in Extension and at what temperature?
Taq DNA polymerase at 72 deg C
What are the seven components of PCR?
Template DNA Nucleotides PCR Buffer Magnesium Chloride Water Forward and Reverse Primers DNA Polymerase
What does heat shock do?
Temporarily open up the cell membrane allowing plasmid DNA to enter the bacteria cell
What are plasmids critical to?
The Human Genome Project
What is Real Time or Quantitative PCR?
The amount of PCR product is measured at the end of each cycle and used to determine the DNA input; most revolutionary since it doesn't wait until the end to be analyzed and gets real time results by measuring florescence
Which codon of the mRNA is positioned to be first read by the ribosome?
The first codon
What is the starting point of recognition in the plasmid?
The origin of replication (ori)
What is selection?
The process of only allowing bacteria with the plasmid to grow by introducing an antibiotic it is resistant to
What are Multiple Cloning sites used for in expression plasmids?
They are the downstream of the promoter and will provide options for getting the gene of interest into the plasmid in the correct direction
What are Multiple Cloning Sites used for in Cloning plasmids?
To open up the plasmid so it is ready to receive a gene of interest. Since there are so many recognition sites, the gene is more likely to find a place to attach to
What are plasmid maps?
Tools used to represent plasmids
What is Electroporation?
Transformation Method that uses electricity to disrupt cell membrane; usually more efficient
Differences between transformation, transduction, and transfection
Transformation: prokaryotes, non-viral methods Transduction: prokaryotes/eukaryotes, viral methods/vectors Transfection: eukaryotes, non-viral methods
What is Reverse Transcription PCR?
Uses the enzyme reverse transcriptase to reverse transcribe mRNA into DNA before PCR begins
What is Nested PCR?
When PCR conditions are not the best, primers may anneal non-specifically and amplify wrong stretches of DNA. So uses a second round of primers
Can plants be genetically modified by plasmids?
Yes
What is the expression of pGLO regulated by?
araBAD operons and AraC protein; will only grow if arabinose is present
What are the two types of genes?
constitutive and facultative
What is a copy number?
number of plasmids per bacterial cell
