CSUN BIOL 107 Lab Exam 1 (Labs 5-8)

wild type

the typical form of a species as it
occurs in nature having no mutations

ADP-1 wild type (wt)

• Succinate = carbon source
• Para-hydroxybenzoate (pHB) = carbon source
• Can metabolize both succinate and pHB
Grow on media containing either succinate
or pHB

mutant

physically different from others of the
same species because of a change in its genes

ADP-6 mutant

• Can only metabolize Succinate

Grow on media containing only Succinate

3 ways bacteria exchange genetic information

1. Transformation
2. Transduction
3. Conjugation

Transformation

● Uptake DNA directly from medium & incorporate it into its genome
● Translocasome in cell envelope uptakes DNA
● Cells must be competent = “willing and able” to be transformed

Take away:

● Bacterium (a single bacteria) will pick up foreign
DNA and incorporate it into
its genome!

How to induce competence

○ Secretes signal (Gram+)
○ Competence factor
○ Stress (i.e. starvation)

Natural Transformation

● Bacteria take up exogenous
DNA on their own
● Naturally occurring
phenomenon

Unnatural Transformation

● Forcing DNA into cells via:
○ Heat Shock
○ Electroporation

Transduction

● Bacteriophage (virus for bacteria) injects DNA
into cell
● Package viral DNA into viral capsid
○ Sometimes accidentally package bacterial
DNA

In other words...

● Bacteriophage (a bacterial virus) will accidentally
pick up bacterial DNA instead of virus DNA and
inject the bacterial DNA into another bacterium.

Conjugation

● “Bacterial Sex”
● Bacteria transfer genetic material via sex pilus
● Pilus proteins encoded on F factor
● Transfers DNA on F factor to recipient cell, which forms new F plasmid
● Lastly, recipient “female” (F-) cells become donor “male” (F+) and can repeat the process

In simpler terms:
● One bacterium, with a sex pilus,
ejects its DNA into a bacterium
with a sex pilus receptor.

What is the purpose of lysis buffer

to resuspend the pellet
(mix up the pellet in solution)

Succinate

• carbon source

• both ADP-1 (wild type) & ADP-6 (mutant) can grow here

Para-hydroxybenzoate (pHB)

• carbon source

• only wildtype ADP-1 can grow here

plasmid

a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan.

Remember: Bacteria have NO nucleus

Plasmid applications

○ Ability to introduce foreign DNA into bacteria allows us to generate additional molecules of DNA – amplification
○ E. coli used in research laboratories – optimal growth in 37°C, asexual reproduction, & growth under normal air conditions
○ Used as mini factories to make many copies of DNA – or to express proteins that can be studied in laboratories

β-lactamase

• enzyme that makes bacteria resistant to the antibiotic ampicillin

• destroys ampicillin structure, disrupting
  its function

• Only plasmids containing this enzyme will be able to grow on media containing ampicillin

Ampicillin

prevents the synthesis of a cell wall – no
cellular replication

green fluorescent protein (GFP)

• Originally from jellyfish, modified for brightness, and encoded on plasmids with ampicillin resistance

• Bacteria will have a plasmid that is either:
  ○ GFP+ → has GFP gene
  ○ GFP- → does not have GFP gene

DNA (Deoxyribonucleic Acid)

• Genetic blueprint found in all living
organisms
• Made up of four bases: A, T, C, and G

A = Adenine

T = Thymine

C = Cytosine

G = Guanine
• Two strands form a double helix
structure

Base Pairs (bp)

• Building blocks of DNA
• DNA sequence

Matching Base Pairs

A ←→ T

C ←→ G

What does it mean for DNA to be Anti parallel?

• the opposite orientations of the two strands of a DNA double helix

• the 5' end of one strand aligns with the 3' end of the other strand

Amino Acids

• Basic units of proteins
• Three-base sequences (codons) in DNA determine specific amino acids
• DNA code determines amino acid sequence

Oligonucleotides

small DNA fragments

dNTPs

free base pairs that are readily available to serve as building blocks during DNA replication and other DNA synthesis reactions

Taq polymerase Enzyme

synthesizes new DNA strands from DNA templates
(produces the complementary strand)

Buffer (no enzyme)

maintains the optimal pH and ionic conditions for enzyme activity, ensuring efficient DNA amplification during the reaction

Polymerase Chain Reaction (PCR) Components

• Oligonucleotides

• dNTPs

• Taq polymerase Enzyme

• Buffer (no enzyme)

PCR steps

1. Denaturation 94°C
2. Annealing ~55°C
3. Extension 72°C

Denaturation

when the double-stranded template DNA is heated to separate it into two single strands

Annealing

when the temperature is lowered to enable the DNA primers to attach to the template DNA

Extension

when the temperature is raised, and the new strand of DNA is made by the Taq polymerase enzyme

Gel Electrophoresis

• Used to separate DNA fragments
based on size (base pairs - bp)
• smaller molecules move faster and
travel farther through the gel
• DNA molecules are negatively charged
and move toward the positive electrode
• Use ‘ladder’ for size reference

Key Plasmid Features

○ Origin of Replication
○ Multiple Cloning Site (MCS)
○ Selectable Marker

Origin of Replication

Necessary for plasmid copying during bacterial replication

Multiple Cloning Site (MCS)

Provides a location for inserting the gene of interest

Selectable Marker

gene encoding a protein that will make the bacteria resistant to an antibiotic or allow growth of the bacteria in the absence of food source