Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

GSBS Biochem > Proteins 2 > Flashcards

Proteins 2 Flashcards

1
Q

questions to ask about your protein

A

disulfides?
exported?
glycosylation?
toxic?
subunits?
detection?

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

MCS

A

multiple cloning site

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

without IPTG, the lac repressor

A

shuts down the T7 polymerase transcription

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

without T7, the gene of interest

A

does not get transcribed and protein does not get made

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

even without IPTG you can get

A

leaky expression

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

there are strains with tighter control of the lac system well suited for

A

toxic proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

components of a plasmid

A

T7 promoter
lac operator
ribosome binding site
his tag
thrombin site
T7 tag
MCS
T7 terminator

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

types of PCR

A

simple
overlapping
quickchange

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

a problem with TAQ polymerase

A

Taq polymerase has a nontemplate-dependent terminal
transferase activity that adds a single deoxyadenosine (A)
to the 3’-end of the PCR products.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

alternative to TAQ

A

PFU

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

in overlapping PCR, that is the linker

A

ser, gly, gly repeats

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

4 steps in typical cloning

A
  1. PCR out GOI using primera with restriction sites
  2. DIgest ends with restriction enzymes and purify
  3. digest expression vector with same enzymes and purify
  4. mix GOI with open vector and add ligase. transform and select via antibiotic resistance
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

three types of ends a restriction digest can create

A

5’ protruding ends
3’ protruding ends
blunt ends

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

3 problems with blunt end cloning

A

religating vector
orientation
low effiecency

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

a cute/cheap trick for blunt end cloning

A

1) Clean the ends of your PCR
with one blunt end enzyme.
2) Open the vector with another
blunt end (here it is SspI)
3) Add SspI to the ligation mix. If
you get empty vector, the
enzyme will cut it open again. If
you get an insert, then the SspI
site is destroyed and stable.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Topoisomerase-based cloning (TOPO cloning) is

A

a molecular biology technique in which DNA fragments
are cloned into specific vectors without the requirement
for DNA ligases.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

The biological role of topoisomerase is to

A

cleave and rejoin supercoiled DNA ends to facilitate
replication.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

The major disadvantage of TOPO® cloning is that

A

very few plasmid backbones are available TOPO® ready, and it
is not feasible to create a TOPO® vector yourself.
Additionally, the efficiency can vary depending on the polymerase used, and the single A overhangs degrade over time, further reducing ligation efficiency. TOPO® ready Gateway® Entry plasmids are also available,
allowing for rapid cloning of PCR products into donor plasmids without the need for restriction enzyme cloning.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

blue/white selection

A

white cells have foreign gene inserted into plasmid.
blue cells have plasmid but not the foreign gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

heat shock transformation

A

uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in
temperature creates pores in the
plasma membrane of the bacteria
and allows for plasmid DNA to enter the bacterial cell

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Electroporation, or
electropermeabilization,

A

is where an electrical field is
applied to cells in order to
increase the permeability of
the cell membrane, allowing
chemicals, drugs, or DNA to
be introduced into the cell.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

quickchange pcr only works if

A

your plasmid is small

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

quickchange pcr uses what to digest the template

A

DpnI

24
Q

The #1 Commandment of cloning and expression:

A

SEQUENCE

25
Q

A possible problem in expression can be

A

that codons commonly used in
animals are often rare in bacteria.
inclusion bodies

26
Q

how to fix rare codon problem

A

rosetta strians or additional vectors

27
Q

advantages of periplasmic expression over cytoplasmic production

A

1: An authentic N terminus can be obtained after removal of the signal sequence by leader
peptidases.
2: The periplasm is conducive to disulfide bond formation because of the presence of the Dsb (thioredoxin family) machinery.
3: There are fewer proteases in the periplasm compared to the cytoplasm and many have specific
substrates.
4: Because the periplasm contains fewer proteins and because its content can be selectively
released by osmotic shock or other strategies purification of the target protein is facilitated.

28
Q

highlights of NEB SHuffle

A

Engineered E. coli K12 to promote disulfide bond formation in the cytoplasm
*Constitutively expresses a chromosomal copy of the disulfide bond isomerase DsbC that promotes the correction of mis-oxidized
proteins into their correct form
*The cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds
*Expresses a chromosomal copy of T7 RNAP
*Tight control of expression by lacI q allows potentially toxic genes to be cloned
*Resistance to phage T1 (fhuA2), Nit, Str, Spec

29
Q

Sometimes, putting a tag on
the protein can help it to

A

fold and/or prevent aggregation -
perhaps by covering up a
greasy spot on the protein.

30
Q

Other ways to improve folding/prevent inclusion bodies

A

decrease the temperature
decrease the induction
Can add 1-3% ethanol or 10-30% glycerol. Both slow down growth and glycerol may help keep the protein soluble.

31
Q

Ecoli pros

A

1.Easy to transform with plasmids
2.Cheap and fast growing
3.Strong and inexpensive expression systems (e.g. IPTG)
4.Wide array of tags and fusion protein options
5.Great for Se-Met and isotope labeling
6.Possible periplasm export for better folding (e.g. pelB, OmpA, DsbA, TolB and MalE)
7.Can co-express with GroELS to improve folding
8.Expression can be controlled with temperature
9.There are expression vectors (e.g. Rosetta) for rare tRNAs not predominant in coli

32
Q

Ecoli cons

A

1.Can form inclusion bodies
2.Often issues with proteins with disulfides
3.No glycosylation or other eukaryotic post translational
modification.

33
Q

ampR

A

ampicillin resistance for bacterial selection

34
Q

AOX1 promotor

A

aldehyde oxidase 1 (for yeast methanol induction)

35
Q

alpha mating factor

A

secretion signmal for export in yeast

36
Q

nourseothricin

A

antibiotic resistance marker for yeast

37
Q

TEF promotor

A

translation effector factor (constitutive)

38
Q

yeast expression can have what kind of colored selection

A

red/white

39
Q

yeast pros

A

1.Easy to transform with plasmids
2.Cheap to grow and fast growth rates
3.Strong and inexpensive expression systems (e.g. MetOH)
4.Wide array of tags and fusion protein options
5.Not bad (not E coli levels) Se-Met and isotope labeling
6.A number of secretion signals with leader processing
7.More like eukaryotic folding
8.Protease deficient strains
9.Glycosylation (but not sialic acid)

40
Q

yeast cons

A

1) Slower to grow and transform than bacteria
2) Harder to crack open the cells than bacteria
3) More expensive to label (i.e. NMR) than bacteria
4) Not quite as many vectors and tools

41
Q

baculovirus expression

A

Ligate gene into ‘donor’ or ‘shuttle’ vector, express vector in bacteria and purify. Transform shuttle vector into special DH10Bac E coli cells that contains the bacmid (a big chunk of the baculovirus genome) and a helper phage to help with the transposition.
With the flanking regions of the shuttle vector, you can get recombination in the bacmid. With the Lac gene, you can select blue/white colonies. Grow them up and purify the bacmid. Add bacmid and helper virus to make full infectious virus in the insect cells. Select, grow, express protein

42
Q

insect cells and baculovirus pros

A

1.Better post-translational modification system
2.Better folding of mammalian proteins.

43
Q

insect cells and baculovirus cons

A

1.Very expensive (media, plasticware, etc).
2.~2 weeks for expression

44
Q

lentivirus expression

A

Lentiviruses (e.g. HIV) can
integrate genes into the genome.
You make your shuttle plasmid
and then co-infect into the
animal cell line. With the help of
the other plasmids, virus is made
and package your gene. Isolate
virus and infect into new cell line.

45
Q

animal cells pros

A

1.Both transient and stable cell line production
2.Very good at making secreted proteins
3.Lentivirus expression systems available for both transient
and stable expression
4.Truly native expression.

46
Q

animal cells cons

A

1.Very time consuming
2.Not cheap

47
Q

two ways to make transgenic plants

A

agrobacterium or gene gun

48
Q

plants pros

A

1.Can make grams of protein
2.No cell culture - seeds and dirt
3.There are some targeted export signals
known

49
Q

plants cons

A

1.Very time consuming
2.Transformation not straightforward
3.If plastid expression, often the same as E. coli with regard to folding problems

50
Q

ways to store proteins

A

1) Precipitate with ammonium sulfate (e.g. 50-60% saturation). Can be stored at 4°C.
2) 10-50% glycerol and frozen at -20°C or -80°C
3) Lyophilized (if you are REALLY lucky)
4) Short term storage with antimicrobials (e.g. azide) and protease inhibitors (e.g. EDTA and protease inhibitor cocktails).
5) Need to know its ‘happy buffer’ (e.g. pH, phosphate versus Tris, and salt concentrations).

51
Q

whats the easiest expression system to use

A

ecoli

52
Q

what expression system might you use for glycosylated proteins

A

anything but ecoli

53
Q

what are the issues with disulfides and how would you fix them

A

wrong disulfide bonds leads to misfolding
periplasm expression
change cell line
change expression system
chaperones

54
Q

how might you deal with eukaryotic proteins that are secreted by the cell

A

flagellar export system
mating export system
golgi
etc

55
Q

what happens when ecoli expresses protein too high

A

inclusion bodies

GSBS Biochem (13 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions