Bacteria, Viruses, Biotechnology

Bacteria (prokaryotes)

living cells

• extract energy from environment and perform variety of metabolic processes to grow, survive, and replicate their genes (made of DNA)

Viruses

non cellular entities

• contain genetic material (DNA or RNA)

• do not have own metabolism, cannot reproduce except within cell of host organism

• All groups of living organisms have their own types of viruses

Capsid

Contain RNA or DNA enclosed within a protein shell

Viruses cannot replicate on their own…

they must enter a host cell

• use hosts cellular machinery (ribosomes, enzymes) to make viral proteins

• the host cell copies the viral genetic material

• new virus particles are assembled and released to infect other cells

• some RNA viruses bring or make special enzymes that help the host cell copy their RNA

Some viruses can be engineered as vectors…

for gene therapy

Gene Therapy

aims to replace, repair, or supplement faulty genes

• scientists insert gene into modified viruses that deliver them to target cells

Retroviruses and other viral vectors integrate new genetic material into the host cells genome…

allowing for long term expression

Properties of Bacteria

Bacterial Chromosomes

Nucleoid

Binary Fission

Plasmids

Bacterial Chromosomes

genes of bacteria are located in it

Nucleoid

chromosome is found there in bacteria (prokaryotes)

• usually a circular shape

Binary Fission

bacteria reproduction, identical copies

Plasmids

small, circular DNA molecules that exist independently of the bacterial chromosome

• occur naturally in bacterial strains and in some eukaryotic cells

• own origin of replication

• they replicate independently of the main chromosome

• not essential for survival but are for growth advantages (antibiotic resistance)

Gene Transfer Between Bacteria

1. Conjugation

2. Transformation

3. Transduction

Genetic Diversity

from mutations or genetic transfer

Conjugation

direct physical interaction

Transformation

DNA released from dead bacterium into the environment is taken up by another bacteria

Transduction

virus transfers genetic information from one bacterium to another

2 major ways to clone DNA

1. vivo cloning

2. vitro cloning (PCR)

Gene Cloning

scientists use bacterial cells as mini factories to make copies of specific genes

Gene copies are used to..

• study gene expression

• create a transgenic organism with disease resistance

• attempt to correct a disease or disorder by supplying a functional gene to a cell with a dysfunctional gene (the hormone insulin)

Insulin Production

DNA cloning using cells

• needed in large quantities for diabetic patients

To produce insulin…

scientists take a copy of human insulin gene and plane it into a bacterial cell. As the bacteria reproduce copies of the insulin gene and its protein product will be made

• use plasmids (recombinant)- bacterial transformation

1st step of gene cloning

cut open plasmid

2nd step of gene cloning

inert DNA sequence of interest from other species

3rd step of gene cloning

reseal the plasmid with recombinant plasmid

4th step of gene cloning

any bacteria that picked up the recombinant plasmid (bacterial transformation) will make more of that sequence every time they divide

This process by which many copies, or clones, of a gene sequence are produced…

is called gene cloning

Restriction enzymes in DNA cloning

enzymes that cut DNA at specific locations

• use these enzymes to cut open plasmids and isolate target DNA sequences

Natural enzymes in bacteria

used for defense against viruses

Restriction enzymes produce cuts with…

cohesive sticky ends

Cohesive sticky ends

can be recombined with other DNA cut with the same enzymes, which will have compatible cohesive ends

DNA Ligase

seals the recombined DNA

How to test if the transformation has worked? The insulin gene is inserted into the plasmid and if bacteria successfully took up the plasmid that contains the insulin gene.

• WHICH bacteria took up the plasmid

• WAS it inserted correctly

WHICH bacteria took up the plasmid

plasmid is engineered to include:

• an antibiotic resistance gene

• the insulin gene

After transformation, the bacteria are spread on agar that contains an antibiotic.

• Survive —> did get the plasmid

• Die —> did not get the plasmid

WAS it inserted correctly

• White colonies = GOOD
  The lacZ gene was disrupted → insulin gene is inserted correctly.

• Blue colonies = BAD
  The lacZ gene is intact → insulin gene did NOT insert.

Polymerase Chain Reaction (PCR)

cloning DNA without cells (vitro)

• process used by scientists to generate a high number of copies of a gene sequence

• way of cloning without using cells

• mimics dna replication

Steps of PCR

repeated cycles of heating and cooling

Practical Applications of DNA Technology:

• Medicine 

• Forensics

• Pharmaceuticals 

• Agriculture 

• Transgenic animals