1. Microbial Genetics and DNA Technology
Viruses
Bacteria
Genetic Engineering
Electrophoresis

2. Viruses Characteristics - not considered alive
- is dependent on another living cell for reproduction

3. Viruses cont’d Structure - noncellular
- has nucleic acid (RNA or DNA) surrounded by a protein coat
- many shapes
- size 500 – 1,000 on tip of pen

6. Two types of viruses
Lytic virus
Lysogenic virus

7. Life Cycle of Lytic Virus
Attaches to cell by tail fibers
Nucleic acid is deposited into cell and attaches to the chromosome of cell
Cell will make new virus parts.
Host cell will eventually lyse (burst) releasing 100’s of virus particles
New viruses invade other cells

9. Types of Lytic viruses Examples
Polio, measles, mumps, colds, flu, chicken pox

10. Lysogenic Virus Life cycle Steps
Inserts nucleic acid into cell.
Viral DNA (prophage) can stay for yrs. and block entry of other viruses.
Host cell can divide and carry with it the viral DNA.
One day the virus becomes activated and goes into the lytic cycle.
Examples HIV, herpes, cold sores

12. Viruses can attack bacteria cells
Virus that attacks the bacteria is called bacteriaphage (phage)
Some bacteria become harmful when they contain the prophage
- botulism, diptheria, scarlet fever

14. Bacteria Prokaryotes Contain a single chromosome usually circular
Some contain an extra circle of DNA called a plasmid which is very useful in genetic engineering.

15. Genetic Engineering

16. Vocabulary Genetic engineering – altering DNA by adding new genes
Plasmids – circular DNA in addition to chromosomes in bacteria
Restriction enzymes – enzymes that cut genes by recognizing specific sequences of nucleotides

17. Phage – virus that attacks a bacteria
Prophage – viral DNA inside a bacterial

18. Recombinant DNA – DNA made from combining 2 different DNA’s
Transgenic organism – the organism made from foreign DNA
Clone – large population of genetically identical organisms made from one original cell

19. Junk DNA – repeats in the genome that don’t code for proteins

20. Steps to Make Transgenic Organism

26. Step 1 Cut plasmid with restriction enzyme
This leaves “sticky ends” that are open and ready to pair with new DNA

27. Step 2 Add new gene to plasmid
This gene has also been cut and has matching “sticky ends”
Result is recombinant DNA

28. Step 3 Mix new plasmid and bacteria w/salt solution and heat shock
This makes bacteria take in the plasmid
New cell called transgenic organism

29. Step 4
Make many copies of the transgenic organism – Clone

30. IV. Uses For Genetic Engineering

31. Engineering in Bacteria
Can make proteins for humans
- Growth hormones
- Insulin

32. Engineering in Plants
Can make complete transgenic plant from recombinant DNA
Goals & uses
- Plant to make own insecticides or nutrients
- Plants to make protein to feed sheep
- Gene for luciferase (enzyme in fireflies) inserted into tobacco plant
-- Plant glows

33. Engineering in Animals
Growth hormones from trout put into carp  bigger fish that grows fast
Genes from HIV given to mice 
used for research

34. Electrophoresis Tool that is used with DNA – see pg.239 Many uses:
1. DNA sequencing
2. DNA fingerprinting

35. DNA Sequencing Reading the order of bases in DNA
Uses only one strand of DNA but many copies of it

36. Steps of Sequencing Make 4 groups of DNA
Treat each with different chemical so that the strand breaks at a certain base
Send samples through electrophoresis
DNA pieces will appear as bands
Pattern of bands reveals the sequence

37. DNA Fingerprinting Techniques used for identifying individuals
Uses “junk DNA”
The number of repeats is different for every person - see board

38. Steps for DNA Fingerprinting
Sm. sample of DNA is cut w/restriction enzyme
Fragments are separated by electrophoresis
Fragments w/repeats are labeled
- shows series of bands
- these bands show length of fragment

39. 4. Pattern of bands are different for every person in the world

41. “Or how I stopped worrying and learned to love the sheep.”
Biotechnology:
“Or how I stopped worrying and learned to love the sheep.”

42. Restriction Enzymes
Restriction enzymes are compounds first isolated in the 1970's
They function by selectively cutting DNA at specific sequences

43. Restriction Enzymes These cuts usually occur in the following forms.
The cut can be made straight across a base-pair sequence resulting in a "Blunt End“
The cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends"
Blunt End
Sticky end

44. Gene Splicing
The presence of sticky ends allows segments of DNA to be joined together. Since DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.

45. Gene Splicing contd..
This allows for genes to be "cut & pasted" between organisms. This can be seen with production of human insulin.
The DNA sequence of insulin is identified and cut out using a restriction enzyme.
A plasmid from E. coli is removed and cut open using the same restriction enzyme
Since both fragments have complimentary sticky ends the bind and the gene for human insulin is integrated into the plasmid
The plasmid is then reinserted into a bacterial cell. This cell will produce insulin and is cultured. Human insulin can now be extracted and provided to diabetics.

47. Gel Electrophoresis
Gel electrophoresis is a technique used to separate fragments of DNA.
Separates fragments as a function of size.
Most types use Agarose to separate fragments.
Agarose is a porous gel. It can allow the passage of molecules through, however, larger molecules move more slowly through it since they cannot squeeze through the pores as easily as smaller molecules.
Electrophoresis Apparatus

48. Electrophoresis Technique
An agarose gel is casted with several holes called wells at one end.
The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution.
Samples of digested DNA are placed in the wells
Electrical leads are attached to the ends of the box creating an electrical potential across the apparatus.
Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus.
Also, since the smaller molecules travel faster through the agarose. Over time this separates the various sized fragments of DNA.
The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA.

49. Finished Gel

50. Gel Electrophoresis

51. DNA Fingerprinting DNA is now a powerful tool in identification.
Based on the fact that the amount of "junk DNA" differs uniquely between individuals.
Structural genes are often separated by large regions of repeating basepairs.
The number of these repeats is unique to an individual.
Therefor when DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to an individual.

52. DNA Fingerprinting Contd…
This will therefor produce a unique banding pattern following a gel electrophoresis.
This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.

53. DNA Fingerprinting

54. Cloning

55. Cloning: What it is
Cloning is the process of making a genetically identical organism through nonsexual means. It has been used for many years to produce plants (even growing a plant from a cutting is a type of cloning). Animal cloning has been the subject of scientific experiments for years, but garnered little attention until the birth of the first cloned mammal in 1997, a sheep named Dolly. Since Dolly, several scientists have cloned other animals, including cows and mice. The recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humans
Dolly, the first mammal clone

56. Dolly: A Mammal Clone Dolly
In 1997, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal.
Wilmut and his colleagues transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe. The nucleus-egg combination was stimulated with electricity to fuse the two and to stimulate cell division. The new cell divided and was placed in the uterus of a blackface ewe to develop. Dolly was born months later.