MCB Lab Practical #4
1.) Show transformed cells (e.coli resistant to AMP) 2.) Normal E.coli growth, (+) control
1.) What is the purpose of using the +pGreen LBA/AMP plate? 2.) What is the purpose of using the +pGreen LBA plate?
1.) no growth, E.coli is sensitive to AMP, (-) control 2.) Normal E.coli growth, (+) control
1.) What is the purpose of using the -pGreen LBA/AMP plate? 2.) What is the purpose ofusing the -pGreen LBA plate?
Ultraviolet (UV) light
A form of non-ionizing radiation that can be used to control microbial growth. Causes the formation of covalent bonds between adjacent pyrimadine bases in DNA
Pyrimidine Dimers
Alter the shape of DNA and lead to mutations, ultimately causing death of the microbe
Amp^r
Ampicillin resistance gene
Genetic Engineering
Bacteria can be engineered to express many different proteins - Medical, environmental, and industrial applications - Horizontal Gene Transfer
Competent Bacteria
Bacteria capable of absorbing naked (free) DNA
Full Damage plate
Bacteria has covered entire plate, both sides look the same
P. aerug because of its biofilm
Based on the results of the zone of inhibition, which bacterium appears to be RESISTANT to most antimicrobials? Why?
S. aureus
Based on the results of the zone of inhibition, which bacterium appears to be SUSCEPTIBLE to most antimicrobials?
Denaturation
Breaking H bond, seperates DNA strand
GFP
Codes for green fluorescent protein
I (intermediate)
Implies clinical efficacy in body sites where the drugs are physiologically concentrated or when a higher than normal dosage of a drug can be used
S (sensitive)
Implies that isolates are not inhibited by the usually achieveable concentrations of the agent with normal dosage schedules
R (resistant)
Implites that isolates are inhibited by the normal concentrations of the drug when the suggested recommended dosage is provided
mecA gene (genotype)
Methicillin resistance; 533bp
Kirby-Bauer Method
Standardized procedure used to determine the susceptibility of bacteria to various antimicrobial agents
Annealing (priming)
Starts replication - DNA primer detects target gene
Extension
Taq polymerase; DNA polymerase which adds nucleotides to elongate DNA strand
Bacterial Transformation
The uptake of naked (free) DNA molecules by competent bacterial cells
Non-ionizing radiation
UV light; excites electrons in molecules. The excitation of electrons in DNA molecules (specifically thymine) in DNA leads to DNA damage
1.) Obtain one 15ml tube and label it -pGreen 2.) Use 100-1000 µl micropipettor and sterile tip to add 250µl of CaCl2 solution to the tube 3.) Place tube on ice 4.) Use sterile loop to transfer one or two large E.coli colonies from LBA culture plate to -pGreen tube 5.) Resuspend cells by repeatedly pipetting in and out 6.) Return the -pGreen tube to ice
What are the steps of Bacterial Transformation for -pGreen tube?
1.) Obtain 3 MHA plates 2.) Label plates: group number, date, name of organism 3.) Swab each plate with S. aureus, E. coli, and P. aerug 4.) Place swab back into paper wrapper and dispose in biohazard 5.) Allow 3-5 min for agar surface to dry before applying disks 6.) Obtain beaker of antimicrobial disks with group number 7.) Sterilize forceps by dipping in ethanol and passing through flame 8.) Place one of each type of disk on each plate using forceps. Keep disk at least 15mm from edge of plate 9.) Apply light pressure to each disk on agar with tip of sterile forceps to secure to medium 10.) Incubate plates for 16-18 hrs at 37 degrees C
What are the steps of Kirby-Bauer Method?
1.) Obtain the number of assigned plates and label bottom of plate with assignment number, name of bacterium, exposure time, and your initials 2.) Use a sterile swab to cover entire surface of agar in each plate with appropiate microbe 3.) Place swab back into wrapper and dispoze in biohazard 4.) Cover one-half of each plate with index card 5.) Place plates under UV hood with lids removed, however, if the assignment number if 8 or 16, the lid is not removed 6.) Expose plates for the correct time durations, incubate at 37 degrees C for 48 hours
What are the steps of UV light lab?
Luria Bertani Agar (LBA) and Luria Bertani Agar with Ampicillin Agar (LBA/AMP)
What are the two agar used in Bacterial Transformation lab?
Staphylococcus aureus and Bacillus megaterium
What are the two bacteria involved in UV light lab?
Kirby-Bauer and PCR
What are the two ways determining antibiotic resistance?
Taq polymerase
What catalyzed the PCR reactions?
Provide nutrition for growth and gene expression
What do LB plates do in Bacterial Transformation?
Neutralize the negative charge which makes them not repel each other
What does CaCl2 do to E.coli and pGreen?
E.coli can transform and become ampicillin resistant and produce green florescent colonies
What does E.coli do when it absorbs the pGreen plasmid?
Opens the porins in outer membrane of E.coli increasing the permeability for the uptake of pGreen
What does Heat Shock do to E.coli and pGreen?
Allows for cell division, where each offspring will carry the new genes and allows bacteria to repair cell walls
What does Recovery (water bath) do in a cell?
CaCl2 + E.coli + pGreen
What does experimental tube contain?
PBP2 (genotype)
What does mecA gene code for?
Inducer
What does the antibiotic act as?
CaCl2 + E.coli
What does the control tube contain?
mecA gene; specific to MRSA strains
What gene are you detecting in PCR lab?
Neutralization
What is CaCl2 used for in Bacterial Transformation?
Escherichia coli
What is an example of competent bacterial cell?
pGreen
What is an example of naked DNA?
1.) DNA (gene) of interest on a plasmid 2.) E.coli strain MM294 3.) CaCl2 4.) Water Bath heat shock 5.) Ice bucket 6.) Luria Bertani Media
What is needed for bacterial transformation?
E.coli MM294
What is the bacteria used in Bacterial Transformation lab?
Plate 7 and 15 has no growth and plate 8 and 16 has substantial growth
What is the difference between plate 7 and 8 of S. aureus and plate 15 and 16 of B. meg?
Recovery
What is the ice bucket used for in Bacterial Transformation?
1.) Plate distribution - LBA/AMP: 100 µl +pGreen - LBA: 100 µl +pGreen - LBA/AMP: 100 µl -pGreen - LBA: 100 µl -pGreen 2.) Use 100-1000µl micropipettor and sterile tip to add 100µl of cell suspension according to the distribution 3.) Sterilize cell spreader and spread cells - Dip in cell spreader in ethanol beaker to sterilize and briefly bass through the Bunsen flame - Lip lid of one plate only enough to allow spreading - Cool spreader by gently rubbing it on the surface of the agar away from cell suspension, and gently drag it back and forth several times across agar surface - Replace lid and dip spreader in ethanol to sterilize 4.) Let plates sit for 3-5 minutes to allow suspension to become absorbed in agar 5.) Incubate plates at 37 degree C for 24-48 hours
What is the plating steps for Bacterial Transformation?
To create a barrier
What is the purpose of covering half of the plate with the index card during the UV light exposure?
Denaturation, Annealing, and Extension
What is the three-step process of PCR?
Induces bacteria to take up DNA
What is water bath heat shock used for in Bacterial Transformation?
Hydrolysis
What process breaks down AMP^r?
millimeters (mm)
What should the diameter of Zone of inhibition be converted to?
Thymine Dimers, creates abnormal bond
What specific mutations or damage does UV light cause to DNA?
RIS table
What table do you refer to, to see the standards of susceptibility of each bacteria to certain drugs?
Amp^r and GFP genes
What two genes does pGreen plasmid contain?
B. meg
Which bacteria (S. aureus/B. meg) is an endospore former?
B. meg, because it has endospores
Which bacterium appears to be more resistant to the effects of UV radiation? Why?
+ pGreen LBA/AMP (experiemental)
Which plate(s) showed transformation?
Non-ionizing
Which radiation can not penetrate barriers?
Ionizing
Which radiation can penetrate barriers?
Amp^r and GFP genes are only expressed in the presence of ampicillin
Why is ampicillin included in the medium?
Ionizing Radiation
X-rays or gamma radiation; carries enough energy to remove electrons from molecules in a cell (ionization)
Zone on Inhibition
Zone where antimicrobial agents diffused into surrounding agar and prevents growth of bacteria
Full damage and no damage plate
Half plate is covered with bacteria, other half has no colonies
Damage and Repair plate
Half plate is covered with bacteria, other half has some colonies
Gel electrophoresis
How can mecA gene be visualized?
Measuring the diameter of zone of inhibition will indicate of an organism is susceptible, resistant, or intermediate
How is susceptibility measured in Kirby-Bauer Method?
2 strands
How many strands of DNA are produced at the end of one cycle of PCR?
Drug resistance
Plasmid exchange and transformation in bacteria occurs in nature as well as clinical settings. What impact does this phenomenon on human disease and healthcare?
Thermocycler
Raises and lowers the temperature of the samples in a holding block in discrete, pre-programmed steps, allowing for denaturation and reanneling samples with various agents
PCR (polymerase chain reaction)
Used to target and amplify target gene; in this lab, mecA
Muellar-Hinton II Agar Plates (MHA)
What agar is used in Kirby-Bauer method?
E.coli, S. aureus, P. aerug
What are the bacteria used in Kirby-Bauer method?
1.) Competent cell prep: CaCl2 2.) Neutralization 3.) Heat shock 4.) Recovery 5.) LB Plating
What are the experimental conditions for Bacterial Transformation?
1.) +pGreen (LBA/AMP) 2.) +pGreen (LBA) 3.) -pGreen (LBA/AMP) 4.) -pGreen (LBA)
What are the four plates at the end of Bacterial Transformation?
1.) Obtain 4 four plates: 2 LBA and 2 LBA/AMP 2.) Label all: - LBA/AMP plate: +pGreen and Experimental - LBA/AMP plate: -pGreen and Negative Control - LBA plate: +pGreen and Positive Control - LBA plate: -pGreen and Positive Control 3.) Heat shock cells in +pGreen and -pGreen tubes. (critical step) 4.) Carry beaker to ice bath and immediately immerse them in 42 degree C water bath for 90 seconds 5.) Immediately return both tubes into ice for 1 min 6.) Allow both tubes to recover in 37 degree C bath for 15-30min 7.) Place both tubes in test tube rack at room temperature 8.) Use 100-100µl micropipettor and sterile tip to add 250µl of LB broth to each tube, gently tap to mix
What are the heat shock steps for Bacterial Transformation?
- Growth: Yes, lawn growth - Color: off-white - Explaination: Normal e.coli growth, no AMP (no inducer)
What are the results of the +pGreen LBA plate, (+) control?
- Growth: Yes, green and white colonies - Color: Green: GFR, transformed cells, White: Satellite cells, nontransformed cells, AMP^r - Explaination: Green: Broke down antibiotic and plasmid is expressed, White: Can't exist w/o green colonies, AMP^r enzyme beta lactamas
What are the results of the +pGreen LBA/AMP plate, experimental?
- Growth: yes, lawn growth - Color: off-white - Explaination: No plasmid (pgreen), normal e.coli growth
What are the results of the -pGreen LBA plate, (+) control?
- Growth: No - Color: N/A - Explaination: e.coli is sensitive to AMP
What are the results of the -pGreen LBA/AMP plate, (-) control?
1.) Obtain one 15ml tube and label it +pGreen 2.) Use 100-1000 µl micropipettor and sterile tip to add 250µl of CaCl2 solution to the tube 3.) Place tube on ice 4.) Use sterile loop to transfer one or two large E.coli colonies from LBA culture plate to +pGreen tube 5.) Resuspend cells by repeatedly pipetting in and out 6.) Return the +pGreen tube to ice 7.) Use 1-10µl micropipettor to add 10µl of pGreen solution into +pGreen tube 8.) Tap tube to mix 9.) Return to ice for 15 mins
What are the steps of Bacterial Transformation for +pGreen tube?
