Quest 3 Flashcards
The process of altering a cell so that it takes up and expresses genetic material acquired from the surrounding environment
Transformation
The state or condition during which bacteria can bind and internalize exogenous DNA molecules, making transformation possible
Competence
Only ____ cells can take up DNA
competent
Bacteria cells can be naturally competent, or can be made competent by artificial means. What are the two artificial methods?
Chemically competent cells - treated with calcium chloride to facilitate attachment of plasmid to bacterial membrane. the cells are heat shocked in a water bath, opening the pores of the cell membrane, allowing the plasmid to enter.
Electrocompetent cells - prepared using electroporation (an electrical pulse creates pores through which DNA enters the cells)
We use ______ cells for most of our transformations because they are efficiently transformed by many plasmids.
DH5alpha
Competent cells need to be handled gently as they are highly sensitive to…
-Changes in temperature
-Mechanical lysis
What will grow on the LB-AMP plate?
Only bacteria resistant to ampicillin will grow on plates
LB-AMP plate contains the same antibiotic that the ________ will provide resistance to.
plasmid of interest
We are using _____ for blue and white colony selection.
X-gal
(With a functional beta-galactosidase present, X-gal will hydrolyze into galactose and a blue dye.)
If a gene is inserted into LacZ, it will disrupt the sequence and the color will be…
white
(LacZ disrupted with insert)
If the LacZ is uninterrupted, the color will be ____.
blue
(LacZ intact = no insert)
When looking at the transformation plates, what are the positive vs. negative controls we will be using?
-The negative control is a blue colony since the color indicates an uninterrupted lacZ gene, so no insert in MCS
-The positive control bacteria that were transformed with a known, confirmed plasmid with calpain insert
What exactly is the positive control?
A positive control is something we know will produce positive results in our assays
What is a negative control that we could have used when setting the transformation?
-Bacteria transformed with an empty pUC19 vector
-Bacteria transformed with a ligation mixture that contained water instead of insert DNA
Bacteria reproduce by a type of cell division called ______.
binary fission
What are the 4 stages for the growth of bacteria?
-Lag phase
-Log (exponential) phase
-Stationary phase
-Death phase
What are the characteristics of the lag phase?
Slow growth, adapting to the conditions
What are the characteristics of the log phase?
Exponential growth - doubling every replication cycle
What are the characteristics of the stationary phase?
Nutrients limiting, waste builds up
Rate of growth = Rate of death
What are the characteristics of the death phase?
Cells die faster than they are replaced
An indirect measure of bacterial growth
Turbidity
A bacterium’s generation time is defined by…
bacterial growth rates under standard nutritional conditions
The generation time for E. coli in the lab is about
17 minutes
Generation time =
time / number of generations
Number of times the cell population doubles during the time interval
number of generations
What factors affect growth?
-temperature
-pH
-salt concentrations
-oxygen levels
-availability of nutrients
How can you troubleshoot a transformation that resulted in no colonies on the plate?
No colonies indicates that the transformation did not work in general
Need to revisit that procedure to determine what went wrong, especially if positive control also had no colonies
How can you troubleshoot a transformation that resulted in all blue colonies?
All blue colonies means no insert
Need to revisit your ligation and possibly RE digestion procedures
How can you troubleshoot a transformation that resulted in a lawn of bacteria (too many total colonies on the plate)?
Restreak from lawn onto a new plate to get isolated colonies
Would all white colonies be a bad thing?
All white colonies could be good because they may all have insert
Could also be bad because it could indicate that the X-gal is not present
Have we successfully cloned our gene?
E. coli have amplified our plasmid in the overnight cultures
We don’t know if it contains our calpain
What is the purpose of making a glycerol stock?
Long-term storage of the bacteria
What are the conditions for a glycerol stock?
-80 degrees celsius and bacteria in 15-20% glycerol
Why do you want to avoid repeated freeze/thaw cycles of glycerol stocks?
It compromises viability
What is the purpose of DNA plasmid stocks?
To preserve bacteria short-term
What are the conditions for DNA plasmid stocks?
4 degrees celsuis - agar plates and liquid culture
What is the purpose of the plasmid purification aka MiniPrep?
To remove RNA, protein, and other cellular components to get purified plasmid DNA
What is a good time to harvest plasmid from bacteria?
In the late log / early stationary phase
How much plasmid we can harvest depends on the…
size of the culture and the kit
Mini kit… up to 20 ug
Midi kit… up to 250 ug
Maxi kit… up to 1 mg
Mega kit… up to 2.5 mg
Giga kit… up to 10 mg
What is the purpose of P1?
Stabilizes pH to 8 to weaken membrane
What is the purpose of P2?
Cell lysis solution: NaOH-SDS
What is the purpose of N3?
Potassium acetate/acetic acid returns pH to neutral (renatures chromosomal DNA into a tangle; chromosomal DNA, proteins, and lipids precipitate out of solution and plasmid DNA remains in solution)
What is the purpose of PB/PE?
Washes off any contaminating salts and SDS attached to the plasmid DNA that remains on the column
What is the purpose of EB?
Elution buffer - removes plasmid from the column
What are the steps to the mini prep?
Bacterial culture is lysed. Lysate is cleared by centrifugation. Lysate is applied to column and plasmid DNA absorbs to the silica gel and impurities are washed away. DNA is eluted in a small volume
We want to verify if the calpain insert is present via
RE mapping
(Calpain insert is flanked by XbaI and PstI restriction sites)
If the calpain insert is present, then if we digest the plasmid with XbaI/PstI, we will be able to see the insert by gel electrophoresis. What size bands do we expect to see?
The 1 kb Calpain insert and the 2.6 kb pUc19 vector.
What goes into a RE digest?
-plasmid DNA
-Reaction buffer 3.1
-XbaI
-PstI
-Water
If the calpain insert is present, then we should be able to amplify it using _____ and the same ____ we used previously and view the insert by gel electrophoresis
PCR; primer
0 dA PCR reaction requires…
-template DNA
-primers
-DNA polymerase
-dNTPs
-thermal cycler
(Gotaq MM contains PCR buffer, dNTPs, MgSO4, and DNA polymerase)
What are the steps to PCR?
- Denaturation - produces single-stranded DNA using heat (95 degrees celsius)
- Annealing - primers bind DNA via complementary base pairing and bracket the sequence to be amplified (50-60 degrees celsius)
- Extension - DNA polymerase adds dNTPs to the 3’ end of each primer (70-75 degrees celsius for 1 min per kb)
Plasmid is an ideal vector for carrying DNA sequences from one organism to another because it is equipped with a
Promoter that enables gene transcription
Sequence for the initiation of DNA replication
Antibiotic resistance gene
Why is it difficult for E. coli to uptake DNA from the environment?
Plasma membrane carefully regulates substances
Cell wall is negatively charged which repels the negatively charged DNA
Why is it important to place the E. coli cells with positive calcium ions?
Neutralizes the negative charge on the cell’s outer membranes, enabling DNA molecules to cross the plasma membranes and enter the cell
What is the purpose of the heat shock?
Sudden increase in temperature causes the pressure outside the cell to increase
The pressure difference enables the plasmid DNA to enter the bacterial cell from the outside
