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Biol 432 > Midterm 2A > Flashcards

Midterm 2A Flashcards

biotech concepts

1
Q

Biotech manufacturing products

A

acronym BRAVABS
Biopolymers (eg Xanthan gum)
REs
AAs (Cyst, Val)
vitamins (eg ascorbic acid Vit C)
Antibiotics
Bioplastics (eg poly-B-hydroxybutyrate)
Small molecs (eg lycopene)

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2
Q

biotech manufacturing - Biopolymers

A

eg Xanthan gum

modify Xanthanomas camestris w/Lac Z and Lac Y genes –> can metabolize lactose or Whey into xanthan
Why? because whey is a really abundant/cheap waste product from cheese industry

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3
Q

biotech manufacturing - REs

A
  • isolated from Ecoli periplasm (space between outter and inner membrane) –> prevent foreign DNA from entering cytoplasm
  • DdeI RE found in same loci as methylase –> recall methylation of DNA prevents self-cleavage
  • place loci on plasmid w/DdeI cut site
  • if loci is inserted –> methylase protects the plasmid –> Ecoli will overexpress DdeI from extra copy
  • if loci fails to insert –> wt DdeI will cleave plasmid therefore degraded
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4
Q

biotech manufacturing - Amino acids

A
  • Serine to cysteine –> controled by negative feedback loop + exces Cyst is degraded. Therefore do mutagenic KO of feedback loop + KO of degradation –> therefore allow mass Cst accumualtion
  • Glucose to valine –> feed backloop control + side rxns w/intermediates (therefore less valine yield) –> KO of side rxns + KO of control regulation + upregulate gluc-val enzymes

basically: KO regulation pathways, KO intermediate side rxns, KO product degradation pathways, upregulate enzymes

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5
Q

biotech manufacturing - vitamins (Reminder: what modifications were made to the transgene?)

A

eg ascorbic acid (vit C)

glucose –> 2,5-DKG –> 2-KLG –> vit C

Erwinia bacteria unable to form 2-KLG –> provide transgenic 2,5-DKG reductase isolated from corynebacterium
- reductase modified w/ Glu192 Arg mutation –> 1.8x stronger activity + 0.75x Km coefficient
- reductase active site gene shuffling –> further 75x stronger activity

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6
Q

biotech manufacturing - Antibiotics

A
  • Most Abs isolated from Streptococcus
  • undecyle prodigiosin - unique reg coloured antibiotic –> easily identified in certain colonies
  • eg polyketide antibiotics
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7
Q

Poly ketide antibiotics

A
  • eg Erythromycin
  • Enzymatic activity comes from 1 massive peptide w/many sites or many peptides working together
  • Features core polyketide synthase backbone –> everything else can be gene shuffled to create new antibiotics
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8
Q

S.aureus and cholesterol

A

SA produces staphyloxanthin using same precursor as human cholesterol
Staphyloxanthin used for ROS detox to protect SA

therefore inhibit precursor synthesis in humans –> SA susceptible to ROS
PT can’t produce cholesterol –> can survive by consuming it in foods

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9
Q

biotech manufacturing - bioplastics - What organism was modified + what additional transgenes required?

A
  • Synthesis of bioplastic by Alcaligenes eutrophs –> slow growing therefore low yield –> need alternative
  • transform various biosynth pathways to Ecoli –> combined transgenic ability to synth bioplastics
  • stabilize plasmid using Par B gene so it is retained in daughter cells
  • provide transgenic fadR + atoC egnes –> ensures enough precursor is made to ensure constant bioplastic synthesis without interruption
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10
Q

Increasing Ethanol production - core principles

A

increased EtOH tolerance
in vivo starch degradation using transgenic amylase + glucoamylase
stronger glucoamylase affinity to allow fermentation of insoluble starch

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11
Q

increasing ethanol production - details

A
  • Recombination of yeast strains + mutagenize SPT15 TF to enhance [EtOH] tolerance
  • transgenic amylase + selection on KanR –> in vivo starch degradation to enhance glucose fermentation (Grow on iodine media –> reacts with starch –> clearing = transformants)
  • transgenic glucoamylase –>in vivo starch breakdown –> integration to genome for transient expression (Modified glucoamylase w/excretion domain + stronger starch affinity –> breakdown insoluble starch)
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12
Q

Pseudomonas superbug for oil degradation

A
  • many strains can degrade various oil xenobiotics –> recombine into a superbug to degrade all of them
  • conjugation with pychrophiles to allow low T degradation
  • Note: Xylene operon - Xylene S and E
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13
Q

Oil degradation - Xyl S

A
  • Xylene S binds substrate to activate the operon –> modify Xyl S to also use/metabolize 4-ethylbenzoate (expanded ease of operon activation
  • Selection using TetR controlled by mutant Xyl S –> grow on tet media w/4-ethylbenzoate –> transformants will use 4-ethylbenzoate to express TetR
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14
Q

Oil degradation - Xyl E

A
  • related to xylene degrdation/metabolism –> can be inhibited by 4-ethylcatechol
  • mutagenize Xyl E so it can’t interact with 4-ethylcatechol
  • selection on 4-ethylbenzoate media in presence of 4-ethylcatechol –> transformants can still metabolize 4-EB in presence of 4-EC
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15
Q

Xenobiotics - Organophosphates - What gene encodes their degradation? how was it modified? effects of modification?

A
  • Ecoli modified with transgenic OPD gene = organophosphate degradation
  • OPD fused with OmpA transmembrane –> enzyme is embedded to CM
  • EC is able to extracelll digest organophosphates –> 7x more activity than cytosolic digestion
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16
Q

Xenobiotics - Nitroaromatics - What enzyme was modified? what are the new nitroaromatic substrates?

A
  • nitroaromatics = carcinogens
  • Burkholderia bacteria carries 4-methyl-5-nitrocatechol monooxygenase
  • unable to natively degrade 4-nitrophenol or 3-methyl-4-nitrophenol
  • error prone PCR to modify 4-methyl-5-nitrocatechol MOase –> able to degrade the new substrates + enhanced degradation of original substrate range
17
Q

xenobiotics - cellulosomes - what is it? what enzymes involved? how to detect these enzymes?

A
  • cellulosomes = protein complexes on surface of microbes carrying cellulolytic domains
  • cell deposits cellulosomes onto plant/fungi –> extracell digestion
  • endoglucanse = initial degradation (detectable by CMC media + congo red stai –> forms yellow spot)
  • exoglucanase = cellulo bi/triose degradation (mAb detection)
  • beta glucosidase= final degradation to glucose
18
Q

transfection by Agrobacterium

A
  • carries Ti plasmid w/T-DNA –> encodes various hormones to induce plant synthesis of food
  • T-DNA is flanked by left/right border sequences
  • T-DNA excised and transfected using Vir proteins

Insert GOI into T-DNA + marker for delivery –> Random integration to host

19
Q

BT pesticide toxin - how it works

A
  • encoded by cry genes
  • Rxn with enzymes in insect gut –> activation –> form gut leakage of ATP and nutrients = death
  • no rxn in human gut –> harmless

recombination with various BT genes = stronger/new toxin

20
Q

BT toxin - transfection method into plants + specific delivery within cell (why there?)

A

agrobacterium using 2 plasmids - 1 carrying GOI (strong P35S promoter) + NPT (KanR), 1 carrying Vir

plasmids have homology region –> recombine to make armed plasmid for actual transfection

Delivery to Chl, NOT nucleus
- chl protein mods similar to bacteria –> easier to make fxnal proteins
- maternally inherited genes (in egg) –> GOI is not HGT’d in pollen

21
Q

cowpea trypsin inhibitor

A

trypsin allows insects to hydrolize plant proteins –> inhibition = death
transfect w/ P35S strong promoter + GOi + NPT (KanR) on 2 Ti plasmids –> recombine into fully armed plasmid

22
Q

Misc pesticides

A

cholesterol oxidase - disrupt midgut membrane
VIP proteins - Gene shuffled to strongest variant (VIP3AcAa) –> transfected with BT to make evolved resistance harder
Cytochrom P450 - neutralizes Gossypol pesticides –> RNAi P450 to make susceptible to Gossypol

23
Q

RNAi - How to impact insects but not the crop?

A

RNAi delivery to chlorophyll –> isolated from nucelus –> no inhibition of cytosolic RNA

injested by insect –> released from chl –> RNAi to insect

24
Q

Plant protection from viruses - what methods?

A

transgenic coat proteins
CRISPR - rolling replication dsDNA can be cleaved by cas9
scFab/FV targetting - recall cancer targetted treatment –> same idea

25
Q

plant protection from viruses - coat proteins

A

Viruses have to decoat capsid to release DNA –> use transgenic coat proteins

mechanism not fully known –> 2 theories:
- transgenic CP causes capsid to reform –> prevent release
- CP transgene forms RNAi to inhibit formation of new virion capsids

26
Q

glyphosate - mechanism

A

inhibits EPSP synthase –> plant can’t produce hormones + AAs

27
Q

resistance to glyphosate

A

N-acetylation (N-acetyltransferase) –> neutralization

insert transgene into plant cell –> can in vivo detox

28
Q

Weeds become glyphosate resistant –> solution?

A

Gene shuffling/error prone PCR to create new glyphosate variants
Requires new N-acetyltransferase to protect plants from new variants

29
Q

Dicambria herbicide

A
  • Mimics auxins –> bind receptors –> prevent auxin uptake
  • resistance by detox into 3,6-dichlorosalicyclic acid form using bacterial dioxygenases
  • transfect dioxygenase –> deliver GOI to chl using transit peptide –> constituitive expression at high [x] (because lots of chl per cell)

Biol 432 (3 decks)

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