What is the Central Dogma?

Try to relate it to today's experiment.

Hint:  ___→___→___→___

The synthesis of new proteins begins with _____ where DNA is used to make RNA, which then produces mRNA.

During DNA _____, the mRNA sequence is converted into amino acids that form proteins.

Briefly explain the semiconservative nature of DNA replication.

1. What are the components that make up a DNA nucleotide?
2. Which nucleotides pair with one another according to complementary base pairing rules?
3. Which nucleotides are pyrimidines, and which are purines?

Hint: nucleotides are adenine, guanine, cytosine, thymine

In the DNA backbone, the deoxyribose sugars are joined at both the __'-hydroxyl and __'-hydroxyl groups to phosphate groups in ester links, also known as "_______" bonds.

What is β-lactamase, and what influence does the gene encoding it have on bacterial growth in certain conditions?

In this lab, we will artificially transform ______ (organism) with the plasmid _____.

The gene for GFP can be switched ON in transformed cells by adding _____ to the cells' nutrient medium.

Transformation efficiency is calculated by dividing the number of ______ by the amount of ______ ____ used for the plate.

Which 3 characteristics of plasmids make them ideal vectors for carrying specific DNA fragments/genes of interest?

_____ are small, circular DNA molecules that exist and replicate independently of the bacterial chromosome.

The pGLO plasmid incorporates a special ____ _____ system to control expression of the fluorescent protein in transformed cells.

_____ encodes a regulatory protein that interacts with the promoter in front of GFP and begins transcription. It is responsive to arabinose.

Cells that are able to take up DNA from the environment are said to be naturally ______.

What is an example organism?

pGLO is regulated under _______ expression. This means that the ampR gene is always ON.

GFP is regulated by araC under ______ expression. This means that it is only ON in the presence of arabinose.

Choices:  constitutive, inducible

What are LB plates, and what are they used for?

What is the purpose of treating the bacteria with CaCl₂?

Why do we use a UV light source to visualize the bacterial colonies after overnight growth?

Why are the agar plates inverted before they are incubated overnight at 37^oC?

Why is it important to use a sterile loop when spreading cells, and being sure to spread the cells evenly across the surface of the agar?

Who coined the term "transformation?"

What organism does the GFP gene come from?

True or False?

Transfection alters the genetic makeup of the recipient cell.

True or False.

The cell must first take up the naked DNA molecule before the DNA can be lysed and fragmented.

True or False.

Amino acids are linked together by phosphodiester bonds.

DNA→RNA→Protein→Trait

Transformed bacteria receive a plasmid DNA containing GFP gene. The gene is coverted into mRNA and the mRNA is then converted into GFP protein. Once GFP is produced, bacteria with the protein have the "glow" trait.

The synthesis of new proteins begins with transcription where DNA is used to make RNA, which then produces mRNA.

During DNA translation, the mRNA sequence is converted into amino acids that form proteins.

Gene expression is the sum of transcription and translation.

DNA replication is considered to be semiconservative because 2 identical copies of all the biological information contained on the original, parental, DNA molecule, are made at the end of replication, but the two new daughter DNA molecules are only "half old" and "half new." Half the original DNA molecule is saved, or conserved in the daughter molecules.

1. Nucleotides are made up of a 5-carbon sugar, a phosphate group, and a nitrogenous base.
2. Adenine pairs with thymine. Guanine pairs with cytosine.
3.  Thymine and cytosine are pyrimidines. Adenine and guanine are purines

Each nucleotide differs only in their nitrogenous base.

In the DNA backbone, the deoxyribose sugars are joined at the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds.

β-lactamase is responsible for resistance to the antibiotic ampicillin. Bacteria carrying the gene for β-lactamase (Amp') will be able to grow in the presence of the ampicillin antibiotic.

In this lab, we will artificially transform E. coli with the plasmid pGLO.

The gene for GFP can be switched ON in transformed cells by adding arabinose to the cells' nutrient medium.

Arabinose is a sugar. Transformed cells will appear white on plates not containing arabinose, and fluorescent green in the presence arabinose.

Transformation efficiency is calculated by dividing the number of transformants by the amount of plasmid DNA used for the plate.

1) Contain an Origin of replication

2) Contain a gene (e.g. antibiotic resistance gene) that allows bacteria carrying the plasmid to be selected

3) Ability to reach a high copy number within bacterial cells.

Plasmids.

The pGLO plasmid incorporates a special gene regulation system to control expression of the fluorescent protein in transformed cells.

araC encodes a regulatory protein that interacts with the promoter in front of GFP and begins transcription. It is responsive to arabinose.

Cells that are able to take up DNA from the environment are said to be naturally competent.

Example: Streptococcus pneumoniae

pGLO is regulated under constitutive expression. This means that the ampR gene is always ON.

GFP is regulated by araC under inducible expression. This means that it is only on in the presence of arabinose.

LB plates are nutrient agar plates containing the necessary nutrients for bacterial growth.

Treating the bacteria with CaCl₂ makes the bacteria competent by preparing the cell wall to become permeable to external sources of DNA. This is generally followed by heat-shocking.

UV light allows visualization of the colonies because of the green fluorescent glow. Bacteria that take up the plasmid should glow green because the plasmid encoded the gene for GFP.

During incubation, condensation forms on the inside of whatever surface is on top. Inverting the plates prevents condensation from dripping onto the bacteria culture.

Using a sterile loop will prevent contamination to the culture. Spreading the cells evenly is necessary to obtain isolated colonies.

Fred Griffith.

He observed that a harmless (rough) strain of S. pneumoniae could be "transformed" into a lethal form if incubated with a heat-killed pathogenic (smooth) strain.



 The GFP gene comes from bioluminescent jellyfish Aequorea victoria. The gene causes the jellyfish to glow and fluoresce green in the dark or under UV light.

True.

False.

The DNA must first be lysed and fragmented before the cell can take up the naked DNA molecule.

False.

Amino acids are linked together by peptide bonds.