L4 genetic engineering of E. coli

Question 1

plasmids, strength and weaknesses

Answer 1

strengths:
- simple
- genome not modified
- maintained by antibiotics
-> plasmid carries resistance gene, E. coli without plasmid will then die because no resistance gene

weaknesses:
- simple: too simple, can’t carry long fragments
- genome not modified: have to knock out one gene to add another
- maintained by antibiotics: not good for industry, expensive

Question 2

transposase

Answer 2

= other way to get gene into genome other than plasmid
- enzyme that binds to end of transposon and catalyzes it to jump in genome
- ex. sleeping beauty
-> recignition site= TA => can go in anywhere
- transposase can make sure gene goes into genome, plasmid can not

Question 3

homologous recombination

Answer 3

• can add genes by exploiting dsDNA break repair
• process: dsDNA break recognized by enzyme=> cuts dsDNA a little bit to travel inbetween => travels to break and cuts so ssDNA “hangs off”
  -> RecA coats ssDNA and tries to locate homologous sequence
  -> with luck the second chromosome has a complementary sequence
  -> inserts ssDNA inbetween dsDNA and “single crossover” event happens
  => recombination, strands get switched

Question 4

why not homologous recombination?

Answer 4

not efficient, cuts don’t happen frequently enough and it would have to happen at the exact right spot.
-> use Crispr-Cas9 instead

Question 5

recombination enzymes from viruses

Answer 5

• very efficient
• process:
  -> have dsDNA with homology regions on each end which are designed based on where in the genome it should go in
  -> virus exonuclease will bind to it and through 5’->3’ activity it’ll create 3’ overhangs
• these will, through enzyme beta, go into the template DNA and thus creating dsDNA with the homology regions on each side

Question 6

recombination enzymes from viruses, the enzymes

Answer 6

beta: single-strand DNA binding protein
alpha: exonuclease
gamma: represses native Rec, from lamda-phages

Question 7

viral integrases

Answer 7

= recombinases
- will add marker to DNA
-> homologous regions on each side of marker => can replace target gene with marker

hm…marker…hm

hm…target…hm (in plasmid)

=> hm…marker…hm (in plasmid)

Question 8

FRT, FLP

Answer 8

• FRT = DNA sequence, repeats
  -FLT= enzyme that recognizes FRT and mistakes it for viral genome => cuts it out
• if add FRT sequence on each side of a gene and FLP is added => gene will be cut out

Question 9

problems and solutions to expressing foreign protein in E. coli

Answer 9

Problems:
- low or high protein amount
- protein not folded correctly
- protein degraded

Solutions:
- alter gene dosage
- control transcription of gene

Question 10

copy-number and control of it

Answer 10

high-copy-number plasmids:
- random partitioning occurs => mutation can lead to one type of cell taking over

low-copy-number plasmids:
- replication coordinated with chromosome replication

can change copy-number by changing ORI => replication more difficult => lower copy-number

Question 11

strong vs weak promoter

Answer 11

• strong = a lot of mRNA produced
• weak = not a lot of mRNA produced

Question 12

tight vs leaky vs constitutive promoter

Answer 12

• tight = needs inducer for transcription to happen
• leaky = will always express some form of activity even without inducer
• constitutive = constant, always on same level

Question 13

pET system

Answer 13

• idea: create strong and tight promoter

1st try:
- added PT7 = viral promoter, transcribed by viral RNAP, not our RNAP
-> it blew through LacI anyway

2nd try:
- added PT7 and viral RNAP, add Lac I in promoter for gene of viral RNAP too
-> tight, both PT7 and RNAP blocked
-> 2 operators = 2 blockings
=> tight!

Question 14

control of translation

Answer 14

• alter RBS => mRNA won’t get translated as well
• “detune” gene
  -> E. coli won’t have all the correct tRNAs straight away