GPAT Biotechnology Syllabus

What is fermentation in the industrial/microbiological sense?

A biochemical process in which microorganisms grown in a bioreactor convert substrates into desired products under controlled conditions (not limited to anaerobic metabolism).

Distinguish batch, fed-batch, and continuous fermentation.

Batch: all nutrients added at start, closed system; Fed-batch: nutrients fed incrementally during the run; Continuous: fresh medium added and product withdrawn continuously at steady state.

What are the typical phases of a microbial batch growth curve?

Lag phase, log (exponential) phase, stationary phase, and death (decline) phase.

What is the difference between trophophase and idiophase?

Trophophase is the growth phase where primary metabolites form; idiophase is the production phase (stationary) where secondary metabolites form.

What is the main difference between submerged and solid-state fermentation?

Submerged fermentation uses microbes growing in a liquid medium; solid-state fermentation grows microbes on a moist solid substrate with little or no free water.

What is an inoculum in fermentation?

The starter culture of actively growing microorganisms added to the production medium to initiate fermentation.

Why is aseptic/sterile technique critical in fermentation, and how is medium usually sterilized?

To prevent contamination that competes with the production strain; medium is typically sterilized by autoclaving (moist heat, 121 C, 15 psi, 15-20 min).

What is downstream processing in fermentation?

The series of steps to recover and purify the desired product from the fermentation broth, e.g., cell separation, extraction, concentration, purification, and formulation.

What is the purpose of a sparger in a bioreactor?

To introduce and distribute sterile air/oxygen as fine bubbles into the medium for aerobic microbial growth.

What does the term kLa represent in bioprocess engineering?

The volumetric oxygen mass transfer coefficient, which quantifies the rate of oxygen transfer from the gas phase to the liquid medium in a bioreactor.

What is a stirred-tank bioreactor and its main functions?

A vessel with an agitator (impeller) that mixes the broth, ensures uniform distribution of nutrients/oxygen/temperature, and keeps cells in suspension.

What is the difference between airlift and stirred-tank bioreactors?

Airlift reactors use injected gas for mixing and aeration (no mechanical stirrer, low shear); stirred-tank reactors use mechanical impellers for agitation.

In bioprocess engineering, what does the specific growth rate (mu) describe, and its equation?

The rate of biomass increase per unit biomass; dX/dt = mu*X, so mu = (1/X)(dX/dt).

What is the Monod equation?

mu = mu_max * S / (Ks + S), relating specific growth rate (mu) to limiting substrate concentration (S), where mu_max is maximum growth rate and Ks is the half-saturation constant.

What is the doubling time formula derived from exponential growth?

td = ln2 / mu = 0.693 / mu, where mu is the specific growth rate.

What is gene cloning?

The process of isolating a specific gene/DNA fragment, inserting it into a vector, and introducing it into a host cell so that multiple identical copies are produced.

What is a cloning vector, and name two common types?

A DNA molecule that carries a foreign gene into a host and replicates within it; examples include plasmids and bacteriophages (also cosmids, BACs, YACs).

What are restriction endonucleases and what do they produce?

Enzymes that cut DNA at specific palindromic recognition sequences; they can produce sticky (cohesive) ends or blunt ends.

What is the role of DNA ligase in cloning?

It joins (seals) the phosphodiester bonds between the insert DNA and the vector DNA, forming recombinant DNA.

What are the essential features of a good cloning vector?

An origin of replication (ori), a selectable marker (e.g., antibiotic resistance), unique restriction sites (multiple cloning site), and small size for easy manipulation.

What is blue-white screening used for in cloning?

To identify recombinant colonies via insertional inactivation of the lacZ gene; recombinants (insert present) appear white, non-recombinants appear blue on X-gal.

What is PCR (Polymerase Chain Reaction)?

An in vitro enzymatic technique to amplify a specific DNA sequence exponentially through repeated cycles of denaturation, annealing, and extension. Invented by Kary Mullis.

What are the three main steps of one PCR cycle and their approximate temperatures?

Denaturation (~94-95 C), annealing of primers (~50-65 C), and extension/elongation (~72 C).

Which enzyme is used in PCR and why is it suitable?

Taq DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus, because it is heat-stable and withstands the high denaturation temperatures.

What is the role of primers in PCR?

Short single-stranded oligonucleotides complementary to the flanking sequences of the target, providing a free 3'-OH end for DNA polymerase to begin synthesis.

What is RT-PCR and how does it differ from standard PCR?

Reverse Transcription PCR first converts RNA into complementary DNA (cDNA) using reverse transcriptase, then amplifies it; standard PCR amplifies DNA directly.

What essential components make up a PCR reaction mixture?

Template DNA, two primers (forward and reverse), Taq DNA polymerase, dNTPs, Mg2+ ions, and buffer.

What is animal cell culture, and what is a primary culture?

Growing animal cells in vitro in a controlled nutrient medium; a primary culture is one started directly from tissue, before the first subculture/passage.

What is a cell line and how does it arise?

A cell population that can be propagated through multiple subcultures (passages); it arises when a primary culture is subcultured for the first time.

Differentiate finite and continuous (immortalized) cell lines.

Finite cell lines have a limited number of divisions then senesce; continuous/immortalized cell lines (e.g., HeLa) can divide indefinitely due to transformation.

What is the difference between adherent and suspension cell cultures?

Adherent (anchorage-dependent) cells grow attached to a surface as a monolayer; suspension cells grow freely floating in the medium.

What is the role of serum (e.g., fetal bovine serum) in cell culture media?

It supplies growth factors, hormones, attachment/spreading factors, and nutrients needed for cell attachment and proliferation.

What is trypsinization in cell culture?

Treatment of adherent cells with the protease trypsin to detach them from the culture surface for subculturing (passaging).

What is contact inhibition in cell culture?

The cessation of cell division and movement when normal cells make contact and form a confluent monolayer; transformed/cancer cells often lose it.

What is a hybridoma and what does it produce?

A cell formed by fusing an antibody-producing B-lymphocyte with a myeloma (cancer) cell; it produces monoclonal antibodies and is immortal.

How is recombinant human insulin produced industrially?

The human insulin gene is cloned and expressed in a host (E. coli or yeast), the protein is produced, then purified; the A and B chains are joined (or proinsulin processed) to form active insulin.

What is Humulin, and why was recombinant insulin a milestone?

Humulin was the first recombinant human insulin (approved 1982), the first FDA-approved genetically engineered therapeutic, replacing animal-derived insulin and avoiding allergic reactions.

How many amino acid chains does insulin have and how are they linked?

Two chains: the A chain (21 amino acids) and B chain (30 amino acids), linked by two interchain disulfide bonds (with one intrachain disulfide in the A chain).

What is a vaccine?

A biological preparation containing an antigen (whole or part of a pathogen) that stimulates the immune system to develop protective immunity and immunological memory against a disease.

Differentiate live attenuated and inactivated (killed) vaccines.

Live attenuated vaccines use weakened but living pathogens (e.g., BCG, oral polio, MMR) giving strong long-lasting immunity; inactivated vaccines use killed pathogens (e.g., Salk polio, rabies) and often need boosters.

What is a recombinant subunit vaccine, with an example?

A vaccine containing a specific antigenic protein produced by recombinant DNA technology rather than the whole pathogen; example: Hepatitis B surface antigen (HBsAg) vaccine made in yeast.

What is the basic principle of an mRNA vaccine?

It delivers messenger RNA encoding a pathogen antigen (e.g., spike protein) into host cells, which then translate it to produce the antigen and trigger an immune response.

What are the main classes of biotechnology by color coding (red, green, white)?

Red = medical/pharmaceutical biotech; Green = agricultural biotech; White = industrial biotech; (Blue = marine/aquatic biotech).

What is a transgenic organism, with a plant example?

An organism carrying a foreign gene from another species; example: Bt cotton, which expresses the Bacillus thuringiensis crystal (Cry) protein for insect resistance.

What is Agrobacterium tumefaciens used for in plant biotechnology?

As a natural genetic engineer; its Ti plasmid transfers T-DNA into plant cells, exploited to introduce desired genes for creating transgenic plants.

What is gene therapy?

A technique that treats or prevents disease by introducing, correcting, or replacing a defective gene in a patient's cells (somatic or, theoretically, germline).

What are monoclonal antibodies and one major application?

Identical antibodies produced from a single B-cell clone (via hybridoma technology) that target a single epitope; used in diagnostics, cancer therapy, and pregnancy test kits.

What is Gardasil, and what does it protect against?

Gardasil is a recombinant HPV (human papillomavirus) vaccine made from virus-like particles (VLPs) of the L1 capsid protein, protecting against cervical cancer-causing HPV strains.