Basics

Question 1

Difference between prokaryotes and eukaryotes (genes)

Answer 1

Prokaryotes: Genetic material not enclosed in membrane

Eukaryotes: Genetic material enclosed in membrane

Question 2

Difference between prokaryotes and eukaryotes (ribosomes)

Answer 2

Prokaryotes: Small, not as many ribosomes

Eukaryotes: Large, numerous ribosomes

Question 3

Difference between prokaryotes and eukaryotes (locomotive)

Answer 3

Prokaryotes: Simple

Eukaryotes: Complex

Question 4

Structural features common to all cells

Answer 4

Cell membrane, cytoplasm, ribosomes,

Question 5

Cytoplasm

Answer 5

Location of organelles where cell perform important functions eg.
protein synthesis, energy production

Question 6

Cell membrane

Answer 6

Regulates the passage of molecules in and out of cells

Question 7

Ribosomes

Answer 7

Protein synthesis

Question 8

Nucleoid

Answer 8

Region within which the genetic material of a bacteria is condensed

Question 9

Nuclear envelope

Answer 9

Membranous structure that protects nucleus and compartmentalizes it from the cytoplasm

Question 10

Cell wall

Answer 10

Provides support and protection for the cell

Question 11

What is the purpose of obtaining a pure culture

Answer 11

For higher yield of products (recombinant proteins). To study a specific strain or species of a cell for their characteristics.

Question 12

Methods of sterilizing medium

Answer 12

Autoclave and membrane filtration

Question 13

DNA

Answer 13

Genetic material can be transcribed by RNA

Question 14

RNA

Answer 14

Encodes for protein in translation

Question 15

Proteins

Answer 15

Macromolecules that carry out cellular functions such as signal relay pathways, cell division and cell adhesion.

Question 16

Peptide bond

Answer 16

The covalent bond between the carboxyl group and the amino group of the neighbouring amino acid

Question 17

Advantage of eukaryote hosts

Answer 17

1. Can produce recombinant proteins that require glycosylation and elaborate folding

Question 18

Advantage of prokaryote hosts

Answer 18

1. Highest growth rate
2. Highest productivity
3. Bacteria media is low cost
4. Bacteria can tolerate the most shear tolerance

Question 19

What is the definition of productivity?

Answer 19

The mass of protein produced, per litre of culture volume in one hour

Question 20

Plasmids

Answer 20

Small, mostly circular, supercoiled DNA molecules.
Contain multiple cloning sites with recognition sequence of many restriction enzymes so that the foreign gene of interest can be inserted. Contains selectable marker (antibiotic resistance gene) that will enable cell with the plasmid to be selected.

Question 21

Cell transformation process

Answer 21

1. Use CaCl2 to make competent cells

2. After incubating with the plasmid at 42C for 2 minutes, the plasmid will be taken up by the cell

Question 22

Primary structure

Answer 22

Polypeptide sequence

Question 23

Secondary structure

Answer 23

Sequences are linked by weak hydrogen bonds to form alpha helices and beta sheets

Question 24

Tertiary structure

Answer 24

The 3 dimensional structure of a polypeptide formed by interactions between R groups of amino acids

Question 25

Quaternary structure

Answer 25

Multiple polypeptides held together by interactions between R groups

Question 26

Why does temperature cause protein to denature

Answer 26

An increase in temperature causes vibrations within the protein molecule, and the energy of these vibrations can disrupt the tertiary structure

Question 27

How can changes in pH cause proteins to denature?

Answer 27

At too high or low pH, some of the charges on the protein R groups will be missing, so the electrostatic interactions that would stabilize the native protein are reduced

Question 28

What is gene expression?

Answer 28

Process by which information encoded in gene is used to synthesize mRNA and protein. It involves transcription, splicing (for eukaryotes) and translation.

Question 29

At the STOP codon UAA, tRNA has a anticodon of AUU

Answer 29

False. There are no tRNAs with anticodons complementary to the STOP codons.

Question 30

How would you perform aseptic transfer of the E. coli glycerol stock culture to the shake flask containing sterile liquid culture medium?

Answer 30

1. Biological safety cabinets should be employed when carrying out the inoculation process 2. Disinfected gloves should be worn at all times and sterile inoculation loops should be used 3. The transfer has to be accomplished quickly and efficiently to minimise contamination of the culture and the environment

Question 31

How would you find out the percentage of cells that has been successfully transformed in a E. Coli culture?

Answer 31

1. The culture needs to be spread to agar plates (without antibiotic) to first develop single colonies 2. Developed single colonies (at least 100) will be then transferred to a new agar plate with antibiotic selection 3. After 24H incubation, the number of colonies developed can be counted (eg. 95 colonies), hence the percentage is 95/100 = 95%

Question 32

Recombinant E. coli cells should never be grown in a medium with antibiotic selection. T or F?

Answer 32

False.

Question 33

A medium with antibiotic selection ensures that recombinant E. Coli cells can retain plasmid DNA. However, cells may delete the foreign gene and thus, do not produce the foreign protein.

Answer 33

True

Question 34

PCR can be used to detect whether a recombinant E. Coli has deleted the foreign gene from plasmid DNA.

Answer 34

True

Question 35

When a protein mixture has the same pH as a protein's pI, the said protein will be precipitated.

Answer 35

True. At the pI, the negative and positive charges are balanced, reducing repulsive electrostatic forces, and the attraction forces predominate, causing aggregation and precipitation.

Question 36

A reporter gene (eg. GFP) can be used to verify whether a recombinant system is able to produce a foreign protein.

Answer 36

True.

Question 37

Antibiotics are heat labile. To sterilise solutions containing antibiotics, autoclaving cannot be used. Membrane filtration should be used instead.

Answer 37

True

Question 38

CHO cells (DHFR-) can survive in cell culture media supplemented with hypoxanthine, aminopterin and thymidine.

Answer 38

True

Question 39

CHO cells (DHFR-) cannot survive in cell culture media containing hypoxanthine and thymidine only

Answer 39

False. They can survive as well.

Question 40

Successfully transfected CHO cells (with DHFR gene) can grow in media containing hypoxanthine and thymidine, but so can unsuccessfully transfected CHO cells (DHFR-)

Answer 40

True

Question 41

Only successfully transfected CHO cells (with DHFR gene) can survive in normal mammalian culture media without hypoxanthine and thymidine.

Answer 41

True

Question 42

MTX can be added to normal mammalian culture media (at low initial amounts) as an inhibitor to the DHFR gene. CHO cells with high copy number of DHFR gene will die, but those with low copy number of DHFR gene will survive.

Answer 42

False. CHO cells with high copy number of DHFR gene will survive despite the MTX concentration.

Question 43

High copy number of DHFR gene does not necessarily mean high copy number of the foreign gene of interest.

Answer 43

False. A high copy number of DHFR gene also means a high copy number of the foreign gene.

Question 44

Differences between prokaryotic cells and eukaryotic cells during transcription and translation.

Answer 44

For prokaryotes, chromosomal DNA not enclosed in nucleus, transcription and translation can happen concurrently, leading to faster gene expression For eukaryotes, splicing occurs between transcription and translation. 5’ cap and poly AAA tail added for greater stability.

Question 45

Explain why prokaryotic cells are not employed as hosts for expression of some eukaryotic proteins.

Answer 45

1. Prokaryotes cannot carry out post-translational processes for protein molecules 2. Some eukaryotic proteins require post-translational process in order to have biological activity, thus prokaryotic cells should not be employed for producing such proteins