Yeast Cloning + Sequencing Flashcards
Which organism did we clone DNA from?
yeast, Saccharomyces cerevisiae
Describe yeast
a unicellular, eukaryotic organism that can reproduce asexually or sexually
What is the number of homologous pairs of chromosomes in haploid yeast cells?
n = 16
What is the number of homologous pairs of chromosomes in diploid yeast cells?
n = 32
Why is yeast a model organism?
small organism
short life cycle
can be grown in large quantities in lab
cheap to grow
grows in liquid and solid medium
contains organelles
does mitosis for asexual reproduction
does meiosis for sexual reproduction
70% of genome is coding region
23% of genome is similar to humans
full genome has been sequenced
What is the overall purpose of this lab?
to clone a fragment of the S. cerevisiae genome into the pUC19 plasmid by creating recombinant DNA and to be sequenced and analyzed to determine which fragment was cloned and its function
What is a cloning vector?
a piece of DNA that can have a small fragment of another foreign DNA fragment inserted into it
What is the cloning vector in this experiment?
pUC19
What is pUC19?
a bacterial plasmid (plasmid University of California #19)
Describe bacterial plasmid
small, circular pieces of DNA that replicate independently from the bacterial genome
Why is pUC19 a useful cloning vector?
because it is a bacterial plasmid, and plasmids allow the isolation, analysis, and manipulation of DNA fragments because they replicate separately from the bacterial genome
What are the 4 major characteristics of pUC19 plasmid that make it useful for this experiment?
presence of:
AmpR gene
ORI
LacZ gene
Multiple cloning site (MCS)
Why is the AmpR gene important in this experiment? which organism has this?
AmpR gene codes for resistance to the antibiotic ampicillin which is used in the media for this experiment
pUC19 has this
Why is the ORI an important quality of pUC19?
the origin of replication allows for independent replication in bacterial host that does not require the entire bacterial genome to replicate - faster replication can occur
Why is the MCS an important quality of pUC19 plasmid?
the multiple cloning site is a region of the plasmid DNA that contains various restriction sites, one of which will be used to cut the circular DNA with a restriction enzyme so that the yeast fragment can be inserted
Why is the lacZ gene an important quality of pUC19 plasmid?
a gene located in the MCS that codes for the beta-galactosidase enzyme which is important for blue-white screening
Which restriction enzyme is used? why?
EcoRI because it recognizes restriction sites in both the pUC19 plasmid DNA and the yeast DNA
What is EcoRI isolated from?
bacteria
What is the purpose of EcoRI in bacteria?
restriction enzymes like EcoRI provide defence for the bacteria against foreign viruses as they can chop them up
How do bacteria protect themselves from being digested by their own restriction enzymes?
bacteria methylate their DNA so the restriction enzyme cannot recognize the restriction site sequences and cut up the genome
What sequence does EcoRI recognize in pUC19?
a 6 bp sequence: 5’ - GAATTC - 3’ located in the MCS
How does EcoRI cut the restriction sequence? What results?
it makes a staggered cut between the G and the A to create sticky, complimentary ends and a linear plasmid
What are the first steps of the project?
to isolate yeast genomic DNA
to digest pUC19 with EcoRI and to dephosphorylate the digested pUC19
What is the purpose of SDS solution (when it is used at any point in this procedure)?
to break down cell walls in yeast
to dissolve cell membranes in bacteria
What is the name of the kit used to isolate the yeast DNA?
GeneJet Genomic DNA Purification Kit
What reagents are included in the GeneJet Genomic DNA Purification Kit?
digestion solution
proteinase K
lysis solution
RNase A
purification column containing silica (not a reagent)
What is the purpose of the digestion solution in the isolation of yeast?
it prepares conditions for digestion
What is the purpose of proteinase K in the isolation of yeast? what kit is it apart of?
proteinase K digests the proteins in the yeast
GeneJet Genomic DNA Purification Kit
What is the purpose of RNase A in the isolation of yeast? What kit is it in?
it removes RNA from the yeast
GeneJet Genomic DNA Purification Kit
What is the purpose of lysis solution in the isolation of yeast? What kit is it in?
ensures the cell is entirely lysed + maintains the pH of required for DNA to bind to the silica membrane of the purification column
GeneJet Genomic DNA Purification Kit
What is ethanol used for while isolating yeast?
ethanol promotes the binding of the yeast DNA to the silica column
What is the purpose of the wash buffers in the GeneJet Genomic DNA purification kit used to isolate yeast?
as the DNA is bound to the silica column, they will wash away impurities
What is the purpose of the elution buffer in the isolation of the yeast DNA?
it allows the yeast DNA to be removed from the silica membrane of the purification column
What restriction enzyme is used to digest pUC19?
EcoRI
Why does pUC19 need to be dephosphorylated after it is digested?
to prevent the rebinding of the cut sticky ends during the ligation reaction
What is used to dephosphorylate pUC19?
alkaline phosphatase
How does alkaline phosphatase dephosphorylate pUC19?
it catalyzes the release of the 5’ phosphate group from the plasmid DNA to prevent cut ends from rebinding during ligation
What procedures were done after the yeast DNA was isolated and the pUC19 DNA was digested and DeP?
yeast DNA was digested with restriction enzyme
competent JM101 E. coli cells prepared
ligation reaction to produce recombinant DNA
transformation and plating
What restriction enzyme was used to digest the yeast DNA?
EcoRI
Why is it important that both the yeast DNA and the pUC19 DNA be cut with the same restriction enzyme?
in order to create sticky ends that are complimentary and that will bind together during ligation to produce recombinant DNA molecules
What organism was used to produce competent cells for transformation?
Escherichia coli
What strand of E. coli was used?
JM101
Why were JM101 E. coli cells used for transformation?
they have AmpS gene (cannot grow in ampicillin)
lack functional lacZ gene
Describe transformation
the process of cells taking up DNA from their environment and incorporating it into their own genome and expressing that DNA
genetic alteration
What does it mean for a cell to be competent? does this occur often in nature?
competent = cell is capable of taking up DNA from its environment (capable of transformation)
uncommon in nature
How were the JM101 E. coli cells made competent?
By exposing them to a chemical solution = T-solution
What are 2 other methods of making cells competent?
- exposing bacteria to ice cold salt and then heat shocking
- electroporation (exposure to electric field to change membrane permeability)
What are the hypotheses for transformation?
DNA molecules pass through large membrane channels in bacterial membrane because:
- exposure to cold salt changes/weakens structure of cell surface
- salt solution contains cations which can reduce/eliminate the repulsion between the negative DNA and membrane phospholipids
- heat shocking produces temperature gradient
What were the two controls used for transforming the competent E. coli cells?
transformation with uncut pUC19
transformation with no pUC19
What is the purpose of transformation?
to make competent E. coli cells uptake recombinant DNA that can be selected and isolated with blue-white screening
What reaction is required to make recombinant DNA?
ligation of the EcoRI digested yeast DNA and pUC19 DNA
What kit was used for the ligation reaction? What major reagent was included in this?
Rapid DNA Ligation Kit
included T4 DNA ligase
What is T4 DNA ligase?
an enzyme isolated from bacteriophage T4 that forms a phosphodiester linkage between the junctions of the hybridized molecules of yeast and pUC19 sticky ends to create recombinant DNA
Why is DNA ligase from T4 bacteriophage used for this experiment?
because it is active at room temperature
What kit was used for transformation? What major reagent was included?
TransformAid Bacterial Transformation Kit
includes T-solution
What is T-solution?
a proprietary solution that induces competency in bacteria cells for transformation
What are the 2 identifiable phenotypes of pUC19?
AmpR gene = can grow in ampicillin
lacZ = produces beta galactosidase which digests XGal in medium in the presence of IPTG to produce a blue pigment in the colonies
What will happen to all the genomes of the JM101 E. coli cells that transform pUC19 plasmid?
any cell that transforms the pUC19 plasmid will convert their AmpS gene to AmpR and be able to grow in ampicillin
What will happen to all the genomes of the JM101 E. coli cells that transform pUC19 ONLY?
AmpS –> AmpR (can grow in ampicillin)
lacZ- –> lacZ+ = will produce B galatosidase and produce blue pigmented colonies
What will happen to all the genomes of the JM101 E. coli cells that transform pUC19 + yeast fragment (recombinant DNA)?
convert AmpS –> AmpR
translation of lacZ+ will not occur because the yeast fragment interrupts the plasmid’s MCS at the location of the lacZ gene = colony grows white
What technique was used to plate the transformed cells?
spread-plate technique under alcohol burner
What were the experimental ligations and what were they plated on?
ligated DeP/EcoRI-digested pUC19 + EcoRI-digested yeast
plated on
LB/Amp/XGAL/IPTG agar plates
What is LB?
luria broth = a medium that contains all essential nutrients for E. coli
What is XGAL?
an artificial substrate of beta-galactosidase that is hydrolyzed if a functional lacZ gene is able to produce B-galactosidase
What is IPTG?
a synthetic analog of lactose which inactivates the lac repressor protein and allows for the synthesis of B-galactosidase
How are IPTG, XGAL, and LacZ gene connected? Why is this important for this lab?
IPTG is a synthetic analog of lactose
a functional lacZ gene produces B-galactosidase only in the presence of lactose of an analog (IPTG)
B-galactosidase is an enzyme that hydrolyzes X-Gal in the medium and precipitates a blue colour and turns the colonies blue
this is important for blue-white screening
What is ampicillin? why is it used in this experiment?
ampicillin is an antibiotic which E. coli cells do not naturally have a resistance to
it is used in this experiment to make sure that any bacterial cells that did not transform the pUC19 plasmid do not grow (no conversion of AmpS –> AmpR)
What were the 3 controls that were plated (including what they were plated on)?
uncut pUC19 on LB/AMP/XGAL/IPTG
no pUC19 on LB only
no pUC19 on LB/AMP/XGAL/IPTG
What was the purpose of the control with uncut pUC19 plated on LB/AMP/XGAL/IPTG? What is the expected result of this control?
E. coli cells that transformed uncut pUC19 were plated as a control to make sure the X-Gal and IPTG in the medium have not degraded and that when produced, B-galactosidase can hydrolyze XGal and turn the colonies blue
expected: only blue colonies
What was the purpose of the control with no pUC19 plated on LB/AMP/XGAL/IPTG? What is the expected result of this control?
E. coli cells that transformed no pUC19 will not be able to grow in the presence of ampicillin (AmpS gene) so this makes sure the ampicillin is working
expected = no growth
What was the purpose of the control with no pUC19 plated on LB only? What is the expected result of this control?
E. coli cells that transformed no pUC19 but were grown on LB only plates makes sure the LB has all the essential nutrients required for the growth of the bacteria
expected = lawn of white colonies
Why would we expect to see a lawn of white colonies in the control with no pUC19 plated on LB only?
Because LB is the complete medium for E. coli so all they need to grow is provided
white because without pUC19, the bacterial cells do not have the lacZ+ gene to produce B-galactosidase, but also XGal and IPTG are not present in the medium so there would be no blue anyways
Why would we expect to see no growth in the control with no pUC19 plated on LB/AMP/XGAL/IPTG?
E. coli cells that did not transform pUC19 will not have been able to convert their AmpS gene to AmpR and will therefore not be able to grow in the presence of ampicillin
Why would we expect to see only blue colonies in the control with uncut pUC19 plated on LB/AMP/XGAL/IPTG?
E. coli cells that transformed uncut pUC19 DNA will have been able to convert their lacZ- gene to functional lacZ+ and in the presence of IPTG, be able to express beta-galactosidase that will hydrolyze the XGal in the medium and precipitate the blue colour, giving the colonies a blue pigment
Why do we expect to see both blue and white colonies in the experimental ligation platings?
because some E. coli cells will transform:
only pUC19 DNA = blue
both pUC19 + yeast DNA = white
those that transform only yeast DNA or no DNA will not grow
Why would E. coli that transformed recombinant DNA appear as white?t
the yeast DNA fragment interrupts the translation of the lacZ+ gene in the plasmid DNA because it is inserted in the MCS of the plasmid DNA (also where the lacZ gene is) so beta-galactosidase cannot be produced and XGal will not be broken down and the colony will not show the blue colour
What is the purpose of blue-white screening?
to determine which colonies of E. coli transformed the recombinant DNA and can be selected for running the gel and sequencing
What is transformation efficiency?
the number of transformant bacterial cells / ug of plasmid DNA
Why are high transformation efficiencies needed?
to maximize the recovery of recombinant DNA molecules
What is the range that transformation efficiencies should ideally be in for successful cloning?
at least 10^6 transformants/ ug of plasmid DNA
What 3 factors effect transformation efficiency?
the amount of DNA used (Saturation can occur)
the form of the DNA (supercoiled is better than linear or single-stranded)
the purity of the DNA
Which plate is used to calculate the transformation efficiency? why?
the uncut pUC19 plate because this DNA is circular and circular transforms better
What is the calculation for transformation efficiency?
= # colonies on uncut pUC19 plate / ng of DNA plated x 1000 ng/ug
How do you calculate the ng of DNA plated?
= pUC19 (ng) / volume of competent cells (uL) x volume plated (uL)
Why were the chosen colonies amplified in LB/Amp liquid media?
Ampicillin used to kill off any bacteria that did not transform the pUC19 DNA
How was the recombinant plasmid DNA isolated from the E.coli cells in LB/Amp liquid?
by Alkaline Lysis Method
Why did we use the alkaline lysis method in this lab?
because it is the preferred technique for plasmid DNA extraction from bacteria
Why is the alkaline lysis method popular?
it is fast and effective
it lyses bacterial cells and destroys genomic DNA while keeping the plasmid DNA intact
Which alkaline lysis kit did we use?
AKA Miniprep
How does the alkaline lysis method isolate the plasmid DNA?
it uses a high pH (~12) to denature large pieces of DNA (the bacterial genomic DNA) which cannot renature in a lower pH and will be precipitated out
plasmid DNA is smaller and can renature when pH is lowered
How are cells lysed in the alkaline lysis method?
SDS dissolves membranes
NaOH provides the basic conditions to break up cell walls and denature the bacterial DNA
Once the cells are lysed, how are the genomic and plasmid DNA separated in the alkaline lysis method?
NaOH provides the basic conditions to denature the DNA (both) but the large genomic DNA cannot renature when the solution is neutralized
when solution is neutralized with KOAc, the genomic DNA and proteins precipitate
What happens during the neutralization reaction in the alkaline lysis method?
KOAc (potassium acetate) is added and the denatured genomic DNA and proteins precipitate out
the plasmid DNA renatures
How is the renatured plasmid DNA precipitated out in the alkaline lysis method?
95% ice-cold ethanol + KOAc precipitate the renatured plasmid DNA
How is the renatured and precipitated plasmid DNA purified completely in the alkaline lysis method?
70% ethanol is used to remove any residual salts
