Bit of a review

What is the point of the exercise?
[R] raw materials
[P] product, including cells and waste

• proteins (amino acids)
• nucleic acids (nucleotides)
• lipids (fatty acids and glycerol)
• polysaccharides (sugars)
• water
• salts

• procaryotes: bacteria, blue-green algae, actinomycetes
• eucaryotes: algae, fungi, yeast, protozoans, plant and animal cells

E.coli

• Genus species or Genus species
• Latin or Greek origins
• Saccharomyces cerevisiae
– saccharo (sugar)
– myces (greek for fungus/yeast)
– cerevisiae (latin for brewer)

• Animal: multicellular, wall-less, ingestive metabolism
• Plant: multicellular, walled, photoautotrophic
• Procaryote: procaryotic cells
• Fungi: eucaryotic, multinucleate with nuclei dispersed in walled and septate mycelum, absorptive metabolism
• Protist: unicellular eucaryotes, single or colonies lacking true tissues, ingestive, absorptive or photoautotrophic metabolism (algae, protozoa, simpler fungi)

Bacillus single cell
(unicellular)
Lampropedia
Streptococcus

cocci rods or baccilli
Borrelia burgdorferi (spiral or spirilla)

• consume alcohol, sugars
• acid-tolerant
• simple organics or complex
• products (eg. Lactic acid)
• fixes nitrogen
• ferments glucose to ethanol

• decaying plants
• soil
• human body
• intestine
• sewage
• specialized environments sewage treatment plant
Escherichia coli

• require oxygen: Bacillus subtilus
• take it or leave it (facultative): E.coli
• obligate anaerobic
– Clostridium botulinim
– Cl. tetani
Clostridium: anaerobic rods with endospores

• Christian Gram, Danish physician, 1883
– smear bacteria on microscope slide
– crystal violet
– iodine solution
– alcohol wash
– safranin counterstain

G+
peptidoglycan (90%) inner membrane
Gouter lipopolysaccharide/ protein membrane peptidoglycan (5-20%)

Gram + or Gram -

4
H 5
OH
CH
2
OH
2
O
1
3 HN
C O
O
CH
3
H
3
4
3
H
5
CH
2
OH
O
1
2
NH
C
C
O
CH
3
O
CH C
L-ala
D-glu
L-lys
D-ala
O
N-Acetyl-D-glucosamine/N-acetylmuramic acid

• treat cells with lysozyme
• osmotically sensitive
• protoplast fusion
• fast grower fused with overproducer gives rise to fast growing producers

• double track membrane
X
O
P O O
-
H
2
C
O
CH
O
C O C O
H
2
CH
2
CH
2
C
CH
2
CH
2
H
2
C
CH
2
CH
2
H
2
C H
2
C
CH
2
CH
2
H
2
C H
2
C
CH
2
CH
H
2
C HC
CH
2
CH
2
H
2
C H
2
C
CH
2
CH
2
H
2
C H
2
C
CH
2
CH
2
H
2
C H
2
C
CH
2
CH
2
H
3
C H
3
C
O
CH
2 phospholipid

• fission
t d
= 10 min possible; 45-60 min common

• budding
t d
= 45 min possible; 90-120 min common bud scar

• mycelial elongation and branching
• surface growth (mats)
• submerged culture
(diffuse mycelia or pellets – 0.1-10 mm)
• some by budding or branching t d
= 60 – 90 min possible; 4-8 h common

Aspergillus
Penicillium

• Actinomyces and
Streptomyces
• procaryotes
• grow as mycelia or pseudomycelia

Bacteria Yeast Fungi
0.5-3
μm
1-5
10 -12 g 10 -11 g
65-75% protein 45-55
15-25% nucleic acid
5-30% carbohydrate/lipid
50-80% water
5-12
10-50
5-15 diameter
25-55
5-10
10-50

Can’t get enough!!!
E.coli composition
• carbon
• oxygen
• nitrogen
• hydrogen
50%
20
14
8
• phosporous 3
• S, K, Na, Ca, Mg, Cl, Fe.... (each 0.2-1%)

• not cells, not living
• not dynamic open systems
• static structure, unable to change or replace its parts
• no respiration, metabolism, biosynthetic fuctions
• subatomic particles containing DNA or RNA
• protein coat
• genetic element, infectious agent

• virus that infect bacteria
• “phage” contamination serious problem in biotechnology
• important vector in genetic engineering

T-2, T-4, T-6 dsDNA phage

HIV virus retrovirus

influenza virus coming out of human cell tobacco mosaic virus herpes virus being assembled in cell nucleus

in vivo
• attached
– connective tissue
– bone
• suspended
– blood
– lymph in vitro
• attachment culture
• anchorage dependent
– microcarriers
– agar
– roller bottles
• suspension culture
– serum
– serum free media

• lymphocytes are leukocytes that grow in lymph tissue
• leukocytes are bacterial or viral phagocytes
• generally difficult or impossible to culture in vitro

• carcinomas, malignant growths
– eg. HeLa cells
• leukemias, malignant growths of blood and lymph tissue
• readily cultured in vitro section of prostate carcinoma

• cervical carcinoma cells
• Henrietta Lacks (1951)
• cultivate virus (vaccines)
• tissue culture studies

Hybridoma (hybrid myeloma) cells fuse spleen β-lymphocyte single antibody mouse or other animal myeloma hybrid myeloma cell produces single molecular species of antibody
(monoclonal)

each lymphocyte produces single antibody (monoclonal)
• diagnostics (peptides, proteins, hormones)
• blood tests (HIV/AIDS)
• affinity adsorbents in bioseparations of antigens

alternative

• raised in rabbits, sheep, goats following injection of antigen
• result is mixture of antibody species from different lymphocytes to different parts of antigen

• HeLa
• HLM
• FS-4
• MK2
• CHO
• L-M
• Sf-9
human human human monkey chinese hamster mouse insect cervix arcinoma fetal liver forskin fibroblast kidney ovary connective tissue
Spodoptera frugiperda

albumine cytokine erythropoietin interleukin 2 insulin monoclonal ab, IgG, IgA human proinsulin chicken hepatocytes transfected monkey fibroblasts baby hamster kidney gibbon lymphoblastoid islets of Langerhans mouse, human/mouse, rat mouse fibroblast

• unspecialized cells that renew themselves through cell division
• under certain physiologic or experimental conditions, induced to becoming differentiated cells

• embryonic
– taken from fertilized 3-5 day embryo (blastocyst)
– can proliferate in vitro for year or more
– genetic and environmental triggers to differentiate
• adult (or somatic) stem cells
– found in bone marrow, muscle, brain...
– undifferentiated cells which generate replacements for lost cells
– cannot proliferate in vitro without differentiating
– differentiate into tissue from which it originates

Embryonic Adult
• pluripotent
• easily grown
• potential for in vitro transplant rejection since donor embryo
• differentiate into tissue of origin
• rare in mature tissues and methods for in vitro cultivation not worked out
• use of patient’s own stem cells avoids rejection

Establishing embryonic stem cell line
• transfer inner cell mass of blastocyst onto nutrient agar
• agar coated with non-dividing mouse embryonic skin cells (feeder layer)
• stem cells grow over plate and subcultured
• long established undifferentiated stem cell line
(many “passages”) called pluripotent
• if cells start to clump together, form embryoid bodies and differentiate spontaneously

• Parkinson’s and Alzheimer’s
• diabetes
• spinal cord injury
• cell degeneration
• muscular dystrophy
• heart disease, stoke
• vision and hearing loss
• burns
• arthritis

• attached callus on solid medium
• aggregated cells in suspension
• plant propogation is key application
Cucumis melo callus protoplasts fusing Brassica protoplasts

• Ginkolides isolated from root, bark, leaves of slow growing tree
• therapeutic agent in control of asthma, alergic reactions and inflammatory reactions, memory
• strong PAF-antagonist
• PAF is platelet activating factor, phospholipid produced by leucocytes, a mediator of inflammatory reactions
• in vitro culture for production is alternative

• somatic embryos
• embryo
• to plantlet