Differentiate between COP I TRANSPORT and COP II TRANSPORT

COP I TRANSPORT :

1. COP I is a protein complex that coats vesicles transporting protein from the cis end of the Golgi complex back to Rough Endoplasmic Reticulum (RER).
2. This type of transport is retrograde.
3. Initiates budding process on cis - end of Golgi complex.
4. Consists of large protein subcomplexes that are made of 7 different protein sub units : Alpha, Beta, Beta', Gamma, Delta, Epsilon and Tau
5. GTPase activity is carried out by ADP Ribosylation Factor (ARF) during the budding process.

COP II TRANSPORT

1. COP II is a protein complex that coats vesicles transporting proteins from the RER to Golgi apparatus.
2. This type of transport is anterograde.
3. Initiates budding process on Rough Endoplasmic Reticulum.
4. Made of 4 different protein subunits. 2 proteins Heterodimers from the coal complex  are -
Sec 23p/ Sec 24p and Sec 13p/ Sec 31p
5. GTPase activity is carried out by GTPase Sar Ip during the budding process.   

Differences between class 1, class 2 and class 3 RNR (Ribonucleotide Reductase)

CLASS 1 RNR:

1. Present in prokaryotes and eukaryotes.
2. Substrate - NDP (Nucleoside diphosphate)
3. Cofactor = Ox bridged binuclear iron center
4. Reductant- Glutaredoxin  and thioredoxin
5. Conditions = functional under aerobic conditions, requires O2 for activation
6. Method of generation of free radicals = the binuclear Fe3+ center interacts with Tyr122 to form the tyrosyl free radical resulting in the generation of a thiol radical (free radical of cys)
7. Exists as alpha2beta2 or alpha2beta6 oligomers.

CLASS 2 RNR:

1. Present in prokaryotes.
2. Substrate = NDP
3. Cofactor= 5-deoxy adenosyl cobalamin
4. Reductant = Glutaredoxin and thioredoxin
5. It is oxygen independent. Can function under both conditions.
6. Radical is generated by homolytic cleavage of the 5-deoxy-adenosyl-cobalamin cofactor. C-Co (III) bond generating a 5-deoxy-adenosyl radical - used to generate thiol radical.
7. Exists only in alpha2beta2 state.

CLASS 3 RNR:

1. Present in anaerobic prokaryotes.
2. Substrate = NTP (nucleoside triphosphate) 
3. Cofactor = [4Fe- 4S] cluster and requires SAM and NADPH for activity
4. Reductant = Provided by oxidation of formate to CO2
5. It is only functional under anaerobic conditions and sensitive to O2.
6. Generated by NADPH supplied and Fe-S cluster mediated one electron reductive cleavage of SAM to yield the 5-deoxy-adenosyl radical which then generates a stable glycyl radical (free radical of glycine , O2 sensitive radical).
7. exists in alpha2 + beta2

Differences between solid-state and submerged fermentation:

SOLID STATE FERMENTATION:

1. WATER ACTIVITY = less
2. NATURE OF SUBSTRATE = insoluble (molasses)
3. GROWTH = Solid state microbes adhere at surface and grow on it.
4. SUBSTRATE = Agricultural waste products are usually used (wheat, gram)
5. MEASUREMENT AND CONTROL OF PARAMETERS = pH, aeration, temperature, agitation and foaming are difficult to measure and control.
6. FLUCTATION IN RESULT = Result vary from batch to batch
7. FERMENTATION TYPE = Only batch fermentation
8. FOAMING = Not a major problem.
9. TYPES OF PRODUCTS = Extracellular product
10. PRODUCT YIELD = Higher concentration of products due to larger amount of substrate available.
11. DOWNSTREAM PROCESSING = Difficult and tedious.
12. STEARILIZATION = It may take long time.
13. EXAMPLES = Harvested product compositing, ripening of cheese, mushroom cultivation, enzyme production etc.

SUBMERGED FERMENTATION:

1. WATER ACTIVITY = more (main component)
2. NATURE OF SUBSTRATE = soluble
3. GROWTH = microbes grow in the suspended form of fungi form mat on the surface
4. SUBSTRATE = make use of crude or synthetic substrates. (crude cheese- whey, molasses)(lactose ,starch growth etc)
5. MEASUREMENT AND CONTROL OF PARAMETERS = pH, aeration, temperature, agitation and foaming are easy to measure and control.
6. FLUCTATION IN RESULT = Result do not vary much from batch to batch
7. FERMENTATION TYPE = Run on batch, fed-batch or continuous 
8. FOAMING =  a major problem.
9. TYPES OF PRODUCTS = can be used for both intracellular and Extracellular product
10. PRODUCT YIELD = Yield is low as the product concentration gets diluted due to large volume of medium used.
11. DOWNSTREAM PROCESSING = Easy recovery of the product
12. STEARILIZATION = It is easy to sterilize. Autoclaving may also be used
13. EXAMPLES = Vitamins production, Amino acids, ethanol, enzyme production.

Differences between continuous and fed-batch

CONTINUOUS:

1. Nutrient addition - Continuously or intermediary with corresponding withdrawal of same volume of media.
2. Type of system - Open system
3. Growth state - Steady state is maintained
4.  specific growth rate = dilution rate 
5. Cell maintenance - It is in log phase.
6. Application - primary metabolites cannot be used for secondary metabolites.
7. catabolite repression - cannot overcome.
8. Types - chemostat, turbidostatic.

FED-BATCH:

1. Nutrient addition - No withdrawal but nutrients or precursors are added without harvesting the medium 
2. Type of system - Partially closed
3. Growth state - A Quasi (pseudo) state is maintained
4.  specific growth rate may not be equal to dilution rate 
5. Cell maintenance - Not necessarily in log phase.
6. Application - Can be used for both primary metabolites and secondary metabolites.
7. catabolite repression - can be overcome.
8. Types - Fixed, variable cyclic

Diauxic growth curve effect of E.coli

Many enzymes are involved in basic cellular housekeeping functions and are synthesized at a more or less constant rate. These are called constitutive enzymes. Other enzymes are synthesized at rates that vary with the cells circumstances and are termed as adaptative or inducible enzymes.

Lactose metabolizing enzymes are inducible:

Bacteria adapt to their environment by producing enzymes that metabolize certain nutrients e.g., lactose, only when these substances are available. E.coli growing in the absence of lactose are initially unable to metabolize this disaccharide to do so, they require the presence of two-protein- beta galactosidase which catalyses the hydrolysis of lactose to its component- monosaccharide and galactosidase permease or lactose permease which transport lactose into the cell. Lactose or one of its metabolic products most somehow trigger the synthesis of above proteins. Such a substance is known as an inducer. The physiological inducer of the lactose system is 1,6 allolactose but isopropyl thiogalactosidase (IPTG) is also a potent inducer which structurally resemble allolactose-lactose system inducers also stimulate the synthesis of thiogalactosidase-trans-acetylase, an enzyme that transfer an acetyl group from acetyl CoA to the 6-OH group of isopropyl-thiogalactosidase such as IPTG.

Lac system genes form an operon- LAC OPERON

The genes specifying wild type beta-galactosidase, lactose permease and thiogalactosidase transacetylase are designated as z, y and a respectively. These are called lac structural genes (genes that specify polypeptide) and are contiguously arranged on E.coli chromosome.

These genes together with the control element 'p and o' form a genetic unit called an operon. Specifically the lac operon. In the absence of inducer, the lac repressor i.e., the regulatory gene prgene product specifically binds tightly to the O gene so as to prevent the transcription of mRNA. On binding inducer, the repressor dissociates from the operator thereby permitting the transcription and subsequent translation of lac enzyme.

Catabolite repression

Glucose is E.coli metabolite of choice the availability of adequate amount of glucose prevents the full expression of genes that encodes protein involved in the fermentation of numerous other catabolites, including lactose, arabinose, galactose, even when these metabolites are present in high concentration. This phenomenon is known as "catabolite repression" which prevents the wasteful duplication of energy producing energy system.

If E.coli grows in a medium containing both glucose and lactose, it uses glucose preferentially until the sugar is exhausted. Then after a short lag, growth resumes with lactose as the carbon source, this biphasic growth pattern or response is called 'diauxic growth'.

cAMP signals the lack of glucose

The greatly diminished level of cAMP in the presence of glucose is the indication that of mechanism of catabolite repression. The increase in cAMP may be due to the effect of the phosphoenolpyruvate phospho-transferase system (PTS) on the adenyl activity, the enzyme that synthesizes cAMP. Enzyme III of PTS denotes a phosphate to glucose during its transport, therefore it enters the cell as glucose-6 phosphate. The phosphorylated form of enzyme III also activates adenyl cyclase.

If glucose is being rapidly transported by PTS the amount of phosphorylated enzyme III is low and the adenyl cyclase is less active, so the cAMP level drops. At least one other mechanism is involved in diauxic growth. When the PTS is actively transported glucose into the cell, non-phosphorylated enz III is more prevalent non-phosphorylated enz III binds to the lactose permease and allosterically inhibits it, thus blocking lactose uptake.

CAP-cAMP complex stimulates transcription of catabolite repressed operons:

CAP is homodimer of 210 residue subunits that undergo a large conformational change on binding cAMP. This CAP is synonymously called as catabolite gene activator protein or cAMP Receptor Protein (CRP).

CAP-(cAMP)2 complex but not CAP itself binds to lac-operon and stimulates transcription from its otherwise low efficiency promoter in the absence of lac repressor. CAP is therefore a positive regulator in contrast to the lac repressor which is a negative regulator (twins off transcription).

Differnces between batch and continuous

BATCH :

1. It is a closed system.
2. All four phases of microbial growth is present.
3. Primary and secondary metabolite are produced.
4. Nutrients are added only one not in between the fermentation process.
5. Process is stopped after the product is formed.
6. Environment conditions inside the batch culture are not constant.

CONTINUOUS:

1. It is an open system.
2. Only lag or exponential phase is present.
3. No secondary metabolite production.
4. Nutrients are added in between the process.
5. Process is not stopped and product is removed.
6. Environment conditions are maintained at constant rate. 

Differences between primary and secondary metabolite:

PRIMARY METABOLITE:

1. Required for the growth and maintenance of cellular functions.
2. Starting substrates of a medium.
3. Consist of vitamins, amino acids, nucleotides etc.
4. Necessary at log phase of microbial growth.
5. Produced to perform physiological functions and support in development of cell.
6. Same in every species means they produce same product.
7. Produced in large quantities.

SECONDARY METABOLITE:

1. Not required for growth.
2. End products of primary metabolite.
3. Consist of antibiotics, steroids, toxins etc.
4. Produced during stationary phase of cell growth.
5. Important in ecological and other activities of the cell.
6. Varies in different species.
7. Produced in small quantities.