BME 3020 Prelim 1 Question and
answers verified to pass
Design criteria - correct answer ✔Functionality, efficiency, tunability,
modularity, durability, etc.
Recombinant DNA - correct answer ✔introducing non-native DNA into
organism
Non-coding regulatory regions - correct answer ✔- Upstream: promoter,
enhancers, repressors, RBS
- Downstream: terminator, regulatory regions
- (EUKARYOTIC) splic out introns, keep only the exons → Open Reading
Frame
Promoter - correct answer ✔- can be inducible or constitutive
- located upstream of ATG (start codon), followed transcription start site (TSS)
Transcriptional differences (PRO and EUK) - correct answer ✔- Polymerase
recognition site/promoter binding site:
• (PRO) -10, -35 upstream of ORF
• (EUK) 50-100 bp upstream
- EUK requires binding of a lot of TFs to recruit RNA polymerase, prokaryotes
don't need
- EUK requires lots of enzymes, heavily regulated
- Location
• (PRO): cytoplasm, (EUK): nucleus)
- Polymerase #
, • (PRO) 1 polymerase, (EUK) 3 polymerases
- (EUK) have TATA box, 5'cap, poly A tail
Terminator - correct answer ✔kicks polymerase off of DNA (on DNA
sequence, but codes for mRNA)
- Bacteria: hairpin (forms in RNA, Rho travels and falls off at hairpin)
- EUK usually have poly-adenlyation, poly A tail at the end
Ribosome Binding Site - correct answer ✔*usually for Prokaryotes only
→ EUK have either a 5'mRNA or IRES (internal ribosome entry site)
Tags - correct answer ✔- Reporter: visualizing, cell sorting, ie. GFP
- Epitope/affinity: purification or fixing protein to a surface
- Solubility: GST, can also help to identify
- Localization tags (ie. SV40)
- Degradation
- Cleavage sites: TEV protease
Tags - Design Considerations - correct answer ✔1) changes shape and size
of protein
2) may need a linker if the tag is large, so that there is no steric hindrance
3) need to remove STOP codon if it is at the C-terminus, or need to remove
the tag's STOP codon if it is tagging protein at N-terminus
4) usually tags monomeric proteins (so it doesn't mess with protein complex)
Ways to clone genes - correct answer ✔- PCR
- Cut and Paste, RE sites
answers verified to pass
Design criteria - correct answer ✔Functionality, efficiency, tunability,
modularity, durability, etc.
Recombinant DNA - correct answer ✔introducing non-native DNA into
organism
Non-coding regulatory regions - correct answer ✔- Upstream: promoter,
enhancers, repressors, RBS
- Downstream: terminator, regulatory regions
- (EUKARYOTIC) splic out introns, keep only the exons → Open Reading
Frame
Promoter - correct answer ✔- can be inducible or constitutive
- located upstream of ATG (start codon), followed transcription start site (TSS)
Transcriptional differences (PRO and EUK) - correct answer ✔- Polymerase
recognition site/promoter binding site:
• (PRO) -10, -35 upstream of ORF
• (EUK) 50-100 bp upstream
- EUK requires binding of a lot of TFs to recruit RNA polymerase, prokaryotes
don't need
- EUK requires lots of enzymes, heavily regulated
- Location
• (PRO): cytoplasm, (EUK): nucleus)
- Polymerase #
, • (PRO) 1 polymerase, (EUK) 3 polymerases
- (EUK) have TATA box, 5'cap, poly A tail
Terminator - correct answer ✔kicks polymerase off of DNA (on DNA
sequence, but codes for mRNA)
- Bacteria: hairpin (forms in RNA, Rho travels and falls off at hairpin)
- EUK usually have poly-adenlyation, poly A tail at the end
Ribosome Binding Site - correct answer ✔*usually for Prokaryotes only
→ EUK have either a 5'mRNA or IRES (internal ribosome entry site)
Tags - correct answer ✔- Reporter: visualizing, cell sorting, ie. GFP
- Epitope/affinity: purification or fixing protein to a surface
- Solubility: GST, can also help to identify
- Localization tags (ie. SV40)
- Degradation
- Cleavage sites: TEV protease
Tags - Design Considerations - correct answer ✔1) changes shape and size
of protein
2) may need a linker if the tag is large, so that there is no steric hindrance
3) need to remove STOP codon if it is at the C-terminus, or need to remove
the tag's STOP codon if it is tagging protein at N-terminus
4) usually tags monomeric proteins (so it doesn't mess with protein complex)
Ways to clone genes - correct answer ✔- PCR
- Cut and Paste, RE sites
