Definitions:
1) What is the simplest definition of biotechnology?
2) What other fields are involved in biotechnology?
3) What is the definition according to European Federation of Biotechnology (EFB)?

2) What was the main reason that influenced the development of Genetic Engineering? (it's not only for plants)
Note: G.E was mainly useful bcuz it improved the superior species production efficiency. ⭐

Core Principles of Biotechnology
i) What are the 2 core techniques that enable biotechnology?
ii) What is rDNA? What is it also called?
iii) What is the importance of bioprocess engineering?

3) Gene Cloning:
i) What is gene cloning?
ii) Why is gene cloning necessary?

First RDT Experiment
1) Who were the 2 dudes who did this experiment and when?
2) What was the aim of their experiment?
3) What kind of vector did they use and from which microbe?
4) Which one did they transfer the rDNA to? (why only to this?)
5) How was the host able to multiply the rDNA?
6) What did molecular scissors refer to?

6) What were the 3 basic steps formulated for G.E an organism?

What are the 3 main tools needed to carry out a genetic experiment? (1st one has 3 subparts)

Overview
1) i) What is the source of R.E's (confusing question)?
ii) Where was it first found and in which year?
iii) What were these enzymes called and what function did each do?

2) i) What is the main function of R.E? (keyword: defined)
ii) What applications can this further serve as?(3)

3) i)Which was the first R.E discovered?
ii) What was the special feature of this R.E?
iii) What do you mean by recognition sequence?
iv) How many other R.E's have been discovered since then and from how many bacterial strains?
v) In which organisms are R.E's only found in? And for what reason? ⭐

4) Naming of R.E's:
i) Explain how the naming process goes (EcoR1). Which organism did it come from (It's more than just E.coli)
ii) Under what category do R.E's come under?
iii) There are 2 types of R.E. What are they and what are their functions?

Working of Restriction Enzymes
1) i) What is unusual nature of recognition sequences that R.E only identify?
ii) Define it. (Key Phrase: orientation of reading is kept straight) (Try and draw it to understand)
iii) What are sticky and blunt ends? What are the other names used for it? Which is the only R.E to give blunt ends?
iv) What is the advantage of sticky ends?

2) i) Elaborate on Mr. Inspector R.E.
ii) Where in the DNA does the R.E cut?

3) V.I.P point about using same R.E for the Plasmid and Host ⭐

1) What is it also known as?
2) i) How does it link the 2 DNA fragments? (like what bonds do it form?
ii) Does this process require energy?

3) i) What is the most used DNA ligase?
ii) Where is it synthesized from?

1) What is it's main function?
2) Is it direction specific?
3) What is the most used DNA Poly?

Step 2: Fragmentation of DNA using R.E's
1) Mention how the R.E enzymes are stored with the DNA for cutting (temp, pH and water)?
2) What is the important point about using same R.E for both stuff?
3) Now after all this cutting we need to separate the desired fragments noe? How do we do this? What is the name of the process?
4) What is the medium in which the DNA is placed?
5) What is the main property of DNA we exploit when using this method?
6) Why do the DNA fragments separate (keyword: sieving effect).
7) What is the purpose of Gel electrophoresis?
8) Now how do we see the DNA fragments? Which chemical is used (intercalating dye)? Can we see it in normal light? What is the colour of the stain?
9) What is the process of taking out the DNA from the gel called?

Step 3: Amplification of foreign DNA.
1) What is this process called?
2) Who invented it?
3) What was the problem they were facing at first?
4) Which organism and enzyme solved this process? What was the main feature of this bacteria?
5) What are the raw material requirements of PCR? (5)

6) Steps in PCR (3)
i) Denaturation:
a) What temp. is required?
b) What happens here?

ii) Annealing:
a) What temp is required here and for how long?
b) What happens here?

iii) Primer Extension (Polymerisation):
a) What temp is needed here?
b) What happens here?

7) Why is PCR so good? (How many copies can we make after 30 cycles?) Give general formula
8) Applications of PCR (2)

Step 1: Extraction/Isolation of DNA
i) What is the first thing we do to increase the bacteria number?
ii) What are the 3 different enzymes used to break the cell wall of bacteria, fungi and plants?
iii) How are the rest of the cytoplasm contents (specifically RNA and proteins) removed?
iv) How is the DNA precipitated?
v) What is this process called?

Step 4: Ligation of DNA fragments

Step 5: Insertion of rDNA into Host
1) How many methods are there?(6)
i) Competency of cell:
1) Why can't DNA pass through cell membranes?
2) How are bacterial cells made competent? (keyword: divalent cations).
3) What happens when they become competent?
4) How is calcium chloride useful?

ii) Transformation:
1) What is the main thing in this method?
2) What temperature range is the bacteria exposed to and for how long?
3) How does this affect the host?

iii) Microinjection
1) What happens here?

iv) Biolistics (from ballistics) and Gene Guns:
1) What is the ammunition?
2) Where is the DNA placed?

v) Disarmed Pathogens vectors:
1) Name the most used ones.

vi) Electroporation:
1) What do they do here? (keyword: short impulse)
2) How does this help the DNA get in (keyword: permeability)

Step 6: Large scale Manufacture
1) What is the ultimate aim of RDT?
2) What is the essential thing needed to make this possible?
2) What is a recombinant protein?
3) What are the 2 main methods of obtaining products in a bioreactor?
a) Batch Fermentation:
i) What do we do here?
ii) What kind of system is this?
b) Continuous Fermentation:
i) What kind of system is this?
ii) What do we do here?
iii) Why is it more advantageous? (keyword: Log phase)
iv) Comment on the biomass quantity produced here.

4) Bioreactors
i) What is a bioreactor?
ii) What are the conditions that are monitored here? (4 + salt and vitamins)
iv) What is an absolute necessity in a bioreactor? (Answer will come soon)
iii) Capacity of bioreactors.
Types of Bioreactors: (2)
i) What is a sparge? What is the advantage of it?
ii) Why are small bubbles preferred over big ones?
iii) What are the control systems present in a bioreactor? (6)

Step 7: Downstreaming process
1) What is this step mainly concerned with?
2)What do they do to the products after purification? (Keyword: Formulate it)