LAB 9 Flashcards
SELECTIVE MARKER
ability to select for/against a phenotype
ex. selecting for antibiotic resistance genes
KANAMYCIN
Antibiotic that inhibits protein synthesis.
Belongs to amino glycoside family
How do cells function normally without Kanamycin?
- Genomic DNA gets transcribed into mRNA.
- Ribosome binds to mRNA.
- Translation occurs and new proteins are made and the cell continues to function normally.
What is the effect of Kanamycin on Kanamycin sensitive E-coli?
Kanamycin enters cells through O2 dependant active transport.
-binds to ribosome and inhibits protein synthesis
What is the effect of Kanamycin on Kanamycin resistant E-coli?
Kanamycin enters cells through O2 dependant active transport.
PHOSPHOTRANSFEREASE phospholyrates Kanamycin, inactivating it.
LIST 3 WAYS ANTIBIOTIC CAN BE TRANSFERRED? (HORIZONTAL TRANSFER) 1) TRANSFORMATION 2)CONJUGATION (PILUS) 3)TRANSDUCTION
TRANSFORMATION
1) antibiotic resistant cell A dies
2) resistance genes (plasmid) is left in the envt.
3) Cell B uptakes the resistance genes and becomes antibiotic resistant.
CONJUGATION (PILUS)
1) cell A transfers plamid using a pilus to cell B
TRANSDUCTION
1)phage infects bacteria and injects DNA/RNA
2)Genetic material is replicated to be packaged into daughter cells
3) very rarely during packaging, do the genomic material get transferred to daughter phage. If bacterial material codes for resistance genes, the phage may transfer this to the next cell it infects.
4)Rather than producing new daughter cells the, the second infected cell will become antibiotic resistant.
LIST THE TREATMENTS THE COMPETENT CELLS WILL UNDERGO
Tube 1: 50uL of Tris buffer and 20 uL of DNAse buffer(no DNA)
Tube 2: 50uL of pDNA and 20 uL of DNAse buffer(no DNA)
Tube 3: 50uL of pDNA and 20 uL of DNAse
What does the TRIS BUFFER do?
maintains pH of 8.
solvent for pDNA
What does the DNA’se buffer do?
- solvent for DNA.
- DOES NOT CONTAIN DNA.
DNA’se
- enzyme that degrades DNA.
- unable to pass through plasma membrane and cell wall. Therefore it should be added to pDNA before the addition of competant cells.
PLATE COUNT METHOD
- viable cell count
- original cell suspension is diluted into suspension of decreasing cell concentration and is spread on a solid medium following incubation.
- following incubation, count the number of colonies that grow on the plate.
VIABLE CELL COUNT
-counts only for living cells because they can grow and divide.
TOTAL CELL COUNT
count both living and dead cells.
2 types of count: Direct and Indirect
PETROFF HAUSSER COUNTING METHOD
- direct cell count.
- microscope slide with a depressed surface and etched slide.
- a thin film of cell suspension of known is spread onto the slide and the number of cells in this volume is directly counted using a microscope.
SPECTROPHOMETERS
- indirect cell count
- turbidity (cloudiness in solution due to presence of particles)
- turbidity is referred as Optical Density
STANDARD CURVE
indirect total count
once you have a standard curve, measuring the culture concentration by the turbidity of cell suspension or concentration of DNA is much easier than performing a direct count using Petroff-Hausser Counting Method.
PERCENT TRANSFORMATION
of competant cells that were transferred/ # of cells that could be transffered * 100
STREAKING
deposit single bacterial cell from liquid medium onto an agar plate
IMPORTANT STEPS IN THIS EXPERIMENT
1) INCUBATE 3 TUBES IN WATER BATH FOR 37 DEGRESS CELSIUS FOR 10 MINUTES
- CACL2 solution
2) INCUBATE TUBES WITH THE COMPETENT CELLS AND THE DNA ON ICE FOR 15 MINUTES
3) TUBES WITH COMPETENT CELLS AND DNA IS PLACED IN 42 DEGREES CELSIUS FOR 30 SECONDS
4) TUBES PLACED BACK ON ICE FOR 5 MINUTES
5) ADDITION OF 500 uL LB MEDIUM ONTO THE TUBES
6) INCUBATE THE HEAT SHOCKED CELLS IN THE 37 DEGREES CELSIUS WATER BATH FOR 20 MINUTES.
1) DNAse added to Tube 3 will degrade the pDNA. DNA’se must be added to DNA before DNA is added to cells as the DNA’se is large and unable to penetrate through the cell membrane of bacterial cell. DNA’se is not taken up by cells like pDNA, therefore, the presence of DNA’se will not affect the genomic DNA located inside cell.
CACL2: creates holes in cell wall and plasma membrane to allow pDNA to enter cells. This also makes cells very susceptible to mechanical damage. It is important to keep cells in ice to prevent the cell from repairing its holes, which will negatively affect the transformation.
2) pDNA enters competent cell. Crosses cell wall, outer membrane, and plasma membrane.
3) HEAT SHOCK: helps pDNA enter competent cells and induces expression of “survival genes” necessary for the repair of damaged plasma membrane(caused by making the cells competent and the heat shock itself.), as well as the overall survival or cells.
4) cell start to repair plasma membrane
5) LB is a standard medium used to grow E-coli.
6) kanamycin resistance genes will be expressed The gene must be transcribed into mRNA and then translated into a polypeptide chain. The protein is phosphotransferase which inactivates kanamycin by phospholyrating it.
What is an inoculum?
culture medium in which microorganisms are grown.
what does the inside of incoluating loop contain?
bacteria and medium
Why should the lid of the agar plate be opened at a 45 degree angle?
to prevent particles from the air to settle on the agar plate.
Touch the loop on the first set of streaks once and scribble the loop on the agar. why?
to pick up portion of cells from the first streak and dilutes/spreads them.
LIST 2 CONTROL GROUPS IN THIS EXPERIMENT
Tube 1: no pDNA
Tube 3: DNAse has destroyed the pDNA.
