Basics Flashcards
Difference between prokaryotes and eukaryotes (genes)
Prokaryotes: Genetic material not enclosed in membrane
Eukaryotes: Genetic material enclosed in membrane
Difference between prokaryotes and eukaryotes (ribosomes)
Prokaryotes: Small, not as many ribosomes
Eukaryotes: Large, numerous ribosomes
Difference between prokaryotes and eukaryotes (locomotive)
Prokaryotes: Simple
Eukaryotes: Complex
Structural features common to all cells
Cell membrane, cytoplasm, ribosomes,
Cytoplasm
Location of organelles where cell perform important functions eg.
protein synthesis, energy production
Cell membrane
Regulates the passage of molecules in and out of cells
Ribosomes
Protein synthesis
Nucleoid
Region within which the genetic material of a bacteria is condensed
Nuclear envelope
Membranous structure that protects nucleus and compartmentalizes it from the cytoplasm
Cell wall
Provides support and protection for the cell
What is the purpose of obtaining a pure culture
For higher yield of products (recombinant proteins). To study a specific strain or species of a cell for their characteristics.
Methods of sterilizing medium
Autoclave and membrane filtration
DNA
Genetic material can be transcribed by RNA
RNA
Encodes for protein in translation
Proteins
Macromolecules that carry out cellular functions such as signal relay pathways, cell division and cell adhesion.
Peptide bond
The covalent bond between the carboxyl group and the amino group of the neighbouring amino acid
Advantage of eukaryote hosts
- Can produce recombinant proteins that require glycosylation and elaborate folding
Advantage of prokaryote hosts
- Highest growth rate
- Highest productivity
- Bacteria media is low cost
- Bacteria can tolerate the most shear tolerance
What is the definition of productivity?
The mass of protein produced, per litre of culture volume in one hour
Plasmids
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.
Cell transformation process
- 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
Primary structure
Polypeptide sequence
Secondary structure
Sequences are linked by weak hydrogen bonds to form alpha helices and beta sheets
Tertiary structure
The 3 dimensional structure of a polypeptide formed by interactions between R groups of amino acids
Quaternary structure
Multiple polypeptides held together by interactions between R groups
Why does temperature cause protein to denature
An increase in temperature causes vibrations within the protein molecule, and the energy of these vibrations can disrupt the tertiary structure
How can changes in pH cause proteins to denature?
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
What is gene expression?
Process by which information encoded in gene is used to synthesize mRNA and protein. It involves transcription, splicing (for eukaryotes) and translation.
At the STOP codon UAA, tRNA has a anticodon of AUU
False. There are no tRNAs with anticodons complementary to the STOP codons.
How would you perform aseptic transfer of the E. coli glycerol stock culture to the shake flask containing sterile liquid culture medium?
- Biological safety cabinets should be employed when carrying out the inoculation
process - Disinfected gloves should be worn at all times and sterile inoculation loops should be
used - The transfer has to be accomplished quickly and efficiently to minimise contamination of the culture and the environment
How would you find out the percentage of cells that has been successfully transformed in a E. Coli culture?
- The culture needs to be spread to agar plates (without antibiotic) to first develop single colonies
- Developed single colonies (at least 100) will be then transferred to a new agar plate with antibiotic selection
- After 24H incubation, the number of colonies developed can be counted (eg. 95 colonies), hence the percentage is 95/100 = 95%
Recombinant E. coli cells should never be grown in a medium with antibiotic selection. T or F?
False.
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.
True
PCR can be used to detect whether a recombinant E. Coli has deleted the foreign gene from plasmid DNA.
True
When a protein mixture has the same pH as a protein’s pI, the said protein will be precipitated.
True. At the pI, the negative and positive charges are balanced, reducing repulsive electrostatic forces, and the attraction forces predominate, causing aggregation and precipitation.
A reporter gene (eg. GFP) can be used to verify whether a recombinant system is able to produce a foreign protein.
True.
Antibiotics are heat labile. To sterilise solutions containing antibiotics, autoclaving cannot be used. Membrane filtration should be used instead.
True
CHO cells (DHFR-) can survive in cell culture media supplemented with hypoxanthine, aminopterin and thymidine.
True
CHO cells (DHFR-) cannot survive in cell culture media containing hypoxanthine and thymidine only
False. They can survive as well.
Successfully transfected CHO cells (with DHFR gene) can grow in media containing hypoxanthine and thymidine, but so can unsuccessfully transfected CHO cells (DHFR-)
True
Only successfully transfected CHO cells (with DHFR gene) can survive in normal mammalian culture media without hypoxanthine and thymidine.
True
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.
False. CHO cells with high copy number of DHFR gene will survive despite the MTX concentration.
High copy number of DHFR gene does not necessarily mean high copy number of the foreign gene of interest.
False. A high copy number of DHFR gene also means a high copy number of the foreign gene.
Differences between prokaryotic cells and eukaryotic cells during transcription and translation.
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.
Explain why prokaryotic cells are not employed as hosts for expression of some
eukaryotic proteins.
- Prokaryotes cannot carry out post-translational processes for protein molecules
- Some eukaryotic proteins require post-translational process in order to
have biological activity, thus prokaryotic cells should not be employed
for producing such proteins
