Acitvating

Question 1

Metagenomics

Answer 1

genes from the environment
extract DNA, sequence
build up a library

Question 2

Asymmetric Chemistry

Answer 2

catalyse one enantiomer

Question 3

Gene Design

Answer 3

consider:
introns, membrane anchor/signal peptide, codon optimisation
homologous: expression in same organism
heterologous: expression I’m different organism

Question 4

Splicing Intron

Answer 4

DNA —> pre-mRNA (transcription)
pre-mRNA —> mRNA (spliced, introns removed)
mRNA —> protein (translation)

Question 5

N-Terminal Anchor

Answer 5

adheres to membrane
cleaned
remove propeptide sequence
remove flanking sequence

Question 6

Codon Optimisation

Answer 6

degenerate
more than one codon for the same amino acid
different organisms use different codons
optimise to the codons which the organism uses

Question 7

PCR

Answer 7

DNA —> single stranded DNA (95C)
ss-DNA + primers —> ss-DNA-primers (55C)
ss-DNA-primers —> copied genes (extend, 72*C)

Question 8

Standard Cloning

Answer 8

plasmid - cut with restriction enzymes
gene from PCR - ligate with linearised plasmid
recombinant plasmid
add antibiotic marker to see recombinant DNA
Histags to purify

Question 9

Ligation Independent Cloning

requires clean up

Answer 9

plasmid
don't need: primer, ATP, DNA ligase 
linearise 
treat T4 dT/A/G/CTP
stops cloning at the specified base
promoters 
PCR 
insert into plasmid 
overhangs anneal

Question 10

InFusion Cloning

no clean up

Answer 10

cut restriction endonuclease 
PCR 
primers
gene extension
digest plasmid 
ligase annuals plasmid

Question 11

Optimise E.coli

Answer 11

IPTG added, low temperature
increased expression
chaperone used to help folding
codon optimisation

Question 12

Pichia

alternative host

Answer 12

AOX1: promote alcohol oxidase, binds RNApol, activate transcription 
alpha-factor: secretion of protein 
MCS: insert gene 
Histags: purify, more soluble 
can modify