Bacterial Cloning Flashcards
what is bacterial cloning?
have bacterial make numerous identical copies of a DNA insert which is put into bacterial plasmid
applications of bacterial cloning?
- biopharmaceuticals
- transgeneic organisms
- cDNA libraries
- genome organization, expression, sequencing
requirements for bacterial cloning?
- competent bacteria (able to take up exogenous DNA)
- appropriate plasmid
- DNA insert
important features of DNA plasmid for cloning?
- origin of replication
- AmpR (ampicilin resistance gene)
- Multiple cloning site
- LacZ operon
what is the difference between normal ori and those found in competent e. coli?
plasmid of interest has a modified version of E.col ori so that we can have multiple plasmids in the same bacteria
this is because when bacteria replicate, each daughter cell needs at least one copy of the plasmid
AND
multiple copies per bacteria = faster expression of DNA insert of interest
What is the AmpR? What is the purpose of this in the experiment?
anitibiotic resistance gene that expresses a protein that confes antibiotic resistance (destroys antibiotic when in cell)
bacteria is put on LB+agar plate with the antibiotic reisitance gene so oly the bacteria with the plasmid survive = SELECTABLE MARKER
if there was no antibiotic resistance gene, what would be seen when bacteria plated?
nothing, antibiotic kills bacteria without plasmid
what is the MCS?
high density of restriction enzyme cut sites area within LacZ operion
this is where the DNA fragment can be inserted/ligated into the plasmid
what does the lacZ operon code for? What does this protein mean for expression?
B-galactosidase protein which creates a blue pigment when it metabolizes a substrate.
substrate is added to LB+agar plates.
gene inactive if DNA insert successfully ligates into lacZ operon, this disrupts B-galactosidase expression = no metabolism = white colonies
gene active if DNA insert doesn’t ligate into lac Z operon, B-galactosidase can metabolize = blue colonies
how is lacZ operon a MARKER GENE?
colonies produced no matter what (bc amp resistance is available) but colonie look difference depending on whether gene is active or not
types of restriction enzymes?
- blunt: slide in fragment
- sticky: offset cut sites, complementarity
characteristics of cut site in MCS?
- specific
- 4-8 BP
- palindromic
for the restriction digest, what is the 3 tubes of testing? What is the components and purpose of each?
- sample of interest: insert tube (add plasmid)
- water tube (add plasmid)
- TE (has TE buffer and no plasmid)
they all have 5x fast digest, water, and EcoRI restriction enzyme
what is the purpose of the H2O tube in the restriction digest?
see if ligation worked correctly (was insert ligated into plasmid properly?)
what is the purpose of the TE tube in the restriction digest?
see if transformation worked correctly (was plasmid inserted into e.coli properly?)
how do you determine how much digest buffer to put in restriction digest?
desired total volume/efficiency of buffer
10µL wanted/5x digest = 2
how do you know how much restriction enzyme to add in each restriction digest?
manufacturer
how do you know how much plasmid/TE tube to add to water and insert tube?
50 ng wanted and 25ng/µL supplied plasmid
add same amount of TE buffer in TE tube
purpose of vortex and centrifuge?
vortex: mix
centrifuge: bring liquid to bottom of tube
double vs single end digest?
both sticky ends
double: two restriction enzymes (ensures certain orientation)
single: one restriction enzyme (insert in either orientation)
how can you ensure that you have complementary ends for sticky ends?
PCR to add corresponding ends
matching restriction enzyme
why do we use both EcoRI and BamHI in our plasmid mapping?
ensure 5’ to 3’ orientation
DOUBLE DIGEST= two diff sticky ends that only complementary one way
what is the insert to vector ratio?
purpose of insert to vector ratio?
molar ration for amount of insert relative to amount of vector/plasmid
normalize based on size (insert or plasmid size affects amt added)
what is the insert to vector ratio calculation?
[ng of plasmid x size of insert x 3 (kbp)]/size of plasmid (kbp)=ng
ng of insert / given insert conc = volume of insert
determine volume of insert needed
what are the experiment tubes in the ligation procedure?
- insert: plasmid already there; add already digested insert
- h2O: plasmid already there
- TE: TE buffer already there
same tubes from restriction digest - cuts both plasmid and insert
all have ligation buffer + DNA ligase + water
why add buffer before ligase?
ligase prefers this bc it is unstable out of ice
what is transformation?
Put exogenous plasmid into E. coli bacteria
why is transformation difficult?
it is difficult to get plasmid DNA into bacteria and pass bacterial membrane
what are the characteristics of the bacterial membrane/plasmic that make it difficult for plasmid to cross through?
- ~400 adhesion zones
- -ve charged plasmid
- -ve charged intracellular space
- -ve charged membrane
what are adhesion zones? what are their effect on transformation?
small gaps in membrane used for interbacterial communication
- too small for plasmid to fit through in its natural state
- they do end up being used for plasmid transformation
what is the effect of -vely charged plasmid on transformation?
plasmid DNA -ve charge due to phosphate backbone = repels bacterial plasma membrane bc it is negative as well
what is the bacterial membrane?
what is the effect of -vely charged bacterial membrane on transformation?
double lipid bilayer
plamid = -ve charge = repel
what is the effect of bacterial intracellular space on plasmid transformation? what controls this effect in the bacteria?
plasmid & intracellular space = -ve
= repel
charge of intracellular space depends to H+ pumps
protocol tries to decrease these barriers of transformation but…
they are still present
What are the 2 phases of transformation?
- competent phase: getting plasmid into bacteria
- lag phase: transcribe, translate, replicate plasmid inside bacteria
how is the -vely charged membrane overcome in the procedure?
what phase is this?
make bacteria COMPETENT with CaCl2
1. Ca2+ and Cl- ions dissociate
2. Ca2+ neutralize neg charge on bact membrane AND plasmid by surrounding both membranes
3. ICE to slow Ca2+ and Cl- molecule movement so neutralization is easy
competent phase
how is the adhesion overcome in the procedure?
phase?
- heat pulse releases lipids from bact. membrane opening pores
- pore + adhesion zones fuse = large opening
- large pores = plasmid enters
+ temperature gradient (hot outside, plasmid wants to leave)
+ osmotic gradient
competent phase
how is the intracellular negative charge overcome in the procedure?
what phase?
- heat pulse SHUTS down H+ PUMPS
- H+ not pumped out = + charge inside bacteria
- plasmid doesn’t repel
competent phase
what are the important components of lag phase?
- LB broth for bacteria to function in lag incubation
- LAG incubation: transcribe, translate, replicate PLASMID in bacteria at bacterial growth optimal temp (transcribe+translate AmpR
why do we do a diluted TE buffer ligation sample?
to see individual colonies present
what are the 5 LB+agar plates?
from restriction + ligation + transformation: insert, H2O, TE
also: plasmid ONLY (no insert) to make sure plasmid amp is working + TE buffer diluted
what is LB broth?
contains nutriens and growth factors for bacteria in lag incubation
what is the dilution solution?
.5% NaCl
how do you do a 10^6 dilution?
10^-2, 10^-4, 10^-6 tubes
each tube gets 990 µL and 10µL of the bacteria from previous tube
this is 10µL of bacteria in 1000µL of solution total which gives a rate of 100 dilution
we can’t do 1µL in 99 of dilution bc we have no way of getting 1µL
