Some products are easy to make, such as proteins or simple molecules which require only one or two steps to make. Other products are hard to make, such as molecules of intermediate complexity that need many genes/proteins.
Alcohol Fermentation
One of the earliest uses of microorganisms to produce something useful or desirable for humans was alcohol fermentation. In this process, glucose is converted to pyruvic acid by glycolysis and the pyruvic acid is subsequently acted upon by pyruvate decarboxylase to yield carbon dioxide and acetaldehyde. It is the acetaldehyde that is converted to ethanol via alcohol dehydrogenase.
Alcohol fermentation in ancient cultures was most often carried out by yeast (a eukaryote), but Zymomonas (a bacterium) was used to produce tequila. Both yeast and Zymomonas both make alcohol rapidly but only from glucose. They cannot use most other sugars and cannot break down starch or cellulose (or other polymers) in order to make alcohol.
Because paper is mostly composed of cellulose, it would be
beneficial to be able to utilize paper waste in the production
of alcohol, especially alcohol for fuel purposes. Cellulose
is a linear polymer of glucose that contains both loosely packed,
amorphous zones and crystalline zones. The amorphous zones are
subject to breakdown first, followed by the crysalline zones.
Cellulose breakdown takes place
in several steps:
1) Endoglucanase snips chains in loosely packed zones
2) Cellobiohydrolase chops off segments of approximately 10 glucoses
3) Exoglucanase cuts off 2 or 3 glucose at a time
4) Cellobiase (b-glucosidase) breaks
it down to single glucose molecules
So one way to efficiently produce alcohol from paper waste would be to transform Zymomonas cells with the cellulose degrading genes via pathway engineering.
Isoenzymes (isozymes) are different forms of an enzyme coded by two (or more) different (but related) genes. (Note: allelic variants are alternative forms of same gene ("allelozymes" or "allozymes"))
Alcohol dehydrogenase (ADH) isoenzyme genes are located
on chromosome 4 in humans. There are three
ADH isozymes that function at different developmental stages
or in different tissues in humans:
a - fetus
b - lung and liver (most important)
g - stomach and liver
Everybody has these three ADH isozymes ( and several other minor ADH's), but there do exist individual variations such that each gene has several different alleles. For example, the b-ADH exists as the b1 allele in 90% of Caucasians, but the b2 allele is the predominant form in most (90%) people of Oriental descent and produces a much more inefficient b-ADH enzyme.
Ice Nucleation Factor
Ice needs a "nucleus" or "seed" to form around. The frost damage of plants is due to ice crystals NOT to the cold itself. The ice nucleation factor INF) is a protein made by some bacteria that can be used as a nucleus for ice formation. (e.g. Pseudomonas syringae, INF present on the outside of the cell) One way to try to prevent frost damage to sensitive crops (such as strawberries) is to prevent bacteria from producing INF. To do this, one might clone the inaZ gene (that encodes INF) into a vector, disrupt the inaZ gene with an antibiotic resistance gene (marker), and then put this construct back into the bacterial cells. This process is called gene knockout or gene disruption.
Blue Genes
The blue/purple dyes of the indigo family are produced naturally in many marine molluscs and were used by ancient cultures (e.g. blue fringe of priests robes in the Bible) and are still used today in blue jeans.
The nah genes are involved in naphthalene breakdown and are found naturally on a plasmid (the NAH plasmid) in several Pseudomonas species.
When some of the nah genes were cloned into E. coli the cell colonies turned blue. This was due to the ultimate formation of indigo from tryptophan because of the combination of two different pathways. Naturally occurring Pseudomonas cannot produce indigo because they lack tryptophanase. However, it is now possible to construct a bioreactor for indigo production.
A bioreactor has several components:
- sterile, closed system
- contains desirable bacterium (either natural or engineered)
- need solid support on which the bacteria will attach
- input: substrate that is used for biosynthesis or a compound
to be degraded (e.g. naphthalene)
- output: synthesized compound or non-toxic breakdown products
(e.g. indigo)
Bioplastics
P = poly
H = hydroxy
A = alkanoate/acid
Chemical structure of PHA plastics
PHB = polyhydroxybutyrate (side chain = -CH3)
PHV = polyhydroxyvalerate (side chain = -CH2CH3)
PHA's are made by some bacteria. They function as energy/carbon storage when the organisms run out of other nutrients (e.g. N). For example, the bacterium Alcaligenes produces mixed PHB/PHV that are licensed as "Biopol" and made by the Zeneca Corporation. This type of plasmtic is currently quite expensive but is completely biodegradable. Therefore, it would be benificial to have a more cost-effective means of making bioplastics. One way would be to put the genes for PHA production in plants and, ultimately, grow them as a "crop."
Arabidoposis is a small plant with a very fast generation time that is often used for genetic studies and has about 10 times as many genes as bacteria. If you put the genes for PHA production into the chloroplast genome, you get 100X better synthesis of PHA than if you put the genes into the nuclear genome. This is because the chloroplast divides independent of the cell and the genes in the chloroplast are translated and transcribed frequently because they are needed for photosynthesis.
a) Proteins (e.g. growth hormone, insulin)
b) Steroids (e.g. sex hormones, corticosteroids)
c) Others (e.g. adrenaline)
Diabetes is, in essence, a lack of insulin and so diabetics must inject insulin to keep blood sugar down to normal. Insulin is a small protein hormone that is secreted by the pancreas. Insulin for diabetics was originally made from the pancreas of cows, but now it is produced via biotechnology and was, in fact, the first genetically engineered protein to be used clinically. (It is sometimes called "humulin")
Insulin is composed of two
polypeptide chains: an A chain of 21 amino acids and a B chain
of 30 amino acids. These two chains are held together by disulfide
bonds formed between cysteine residues.
There is a problem when producing insulin in bacteria. If the insulin gene is cloned into bacteria, preproinsulin and NOT insulin is produced. The preproinsulin must be processed to produce insulin so the bacteria also need the genes for the processing enzymes in order to make insulin. In practice, you make two artificial mini-genes - one for A-chain and other for B-chain.
Approach for "cloning" insulin
a) chemical synthesis of DNA for A-chain
b) insert into plasmid next to a bacterial promoter
c) transform into E. coli
d) bacteria grow and make lots of insulin A-chain
e) break open bacteria and purify the A-chain
f) do the same for B-chain
g) mix A and B - oxidize to form disulfide bonds
Step (g) is the snag in this procedure because you actually get a mixture of linkages which results in a 10-20% yield. But in real life, the two chains are synthesized from artificial mini-genes fused to b-galeactosidase. Why is it done this way? Because small insulin chains would not fold on their own, therefore they would be degraded by bacteria. Natural insulin clumps, forming dimers and hexamers, and the surface which binds to the insulin receptor is hidden in the hexamer. Improved insulin is genetically engineered to stop dimer and hexamer formation . Engineered insulin has an aspartic acid residue as opposed to a proline residue so the negative charges of the aspartic acid repel each other and prevent hexamer formation.
Fat
35 million Americans are fat enough to seriously affect their
health
Homozygous ob-/ob- (ob = obese gene) mice are very fat. However, if you provide leptin to these obese mice, they eat less and burn off 30% of their body weight (fat). Mouse leptin and human leptin genes have both been cloned into E. coli and they both function properly.
Several known genetic defects affect how fat you are. If someone is obese and has been from birth, either the leptin gene (ob) or the leptin receptor gene (db) are nonfunctional. If someone grows fat with age, this may indicate a problem with the fa gene that results in insulin not being properly processed and, thus, proinsulin builds up in the body. If someone is simply tubby (not obese), it may be due to genetic factors but, more likely, is due to overeating and/or eating the wrong types of food. It is also interesting to note that some people who are severely overweight were infected with a virus, Adenovirus-36.
The hypothalmus secretes neuropeptide Y (NPY), which increases feeding and makes you fatter. Leptin from the fat cells travels through the blood to the hypothalmus where it is recognized by the leptin receptor. Binding of leptin to the leptin receptor triggers the hypothalmus to stop production of NPY.
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Last updated: 21-Mar-99 / laa
