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