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BIOMEDICAL ENGINEERING & OMICS BACTERIAL DNA & LAB 1; LAB SAFETY & NOTEBOOKS Dawn M. Kilkenny, PhD January 9th, 2013 Institute of Biomaterials andBiomedical Engineering

Bacteria as a Bioengineering Tool n Mammalian genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use (i.e., human insulin, human growth hormone, vaccines, etc.) n Genes from one organism can be transcribed and translated when put into another kind of organism (individuals receiving
genes from another species are referred to as "transgenic")

Expediency of Bacterial Growth Bacterial Plasmid DNA n Most commonly found as small circular, double-stranded DNA molecules in bacteria.
n Small DNA molecules that are separate from, and can replicate independently of, bacterial chromosomal DNA within a cell. n Plasmids carry their own genes for specialized functions.
n Can be genetically altered (engineered) so that if taken up by bacterial cells, the cells will carry out a specific function. n Plasmids used in genetic engineering are called vectors.
n Commonly used to make multiple copies of, or introduce expression of, specific genes of interest.
n The gene to be replicated is inserted into the multiple cloning site (MCS) of a plasmid that already contains genes that make the cells resistant to specific antibiotics .
n The MCS (polylinker) is a short nucleotide sequence containing common enzymatic restriction sites allowing easy insertion of DNA fragments at this location. Plasmid DNA Replication Plasmid replicates with the bacterial cell and can be transmitted from cell to cell.
Plasmids ensure a DNA exchange, and are responsiblefor bacterial adaptation and survival.
Plasmids can be considered
part of the mobilome
because often associated
with conjugation (a
mechanism of horizontal
gene transfer).
n The process by which foreign DNA (i.e., from the environment) is introduced into a bacterial cell.
n Acquisition of new genetic material can provide living bacteria with survival advantages.
n Transformation in nature is rare however researchers have developed experimental techniques to introduce plasmids into bacterial cells.
n Bacterial strains have been created in the biomedical research lab that can easily be transformed (i.e., DH5α competent cells) Transformation at the Molecular Level n In order for a bacterium to take in foreign DNA, the plasmid must pass through the bacterium's cell wall and plasma membrane.
n To facilitate this, researchers use a chemical treatment to make the cells "competent" (capable of taking up foreign DNA).
n The process alters the bacteria's plasma membranes in such a way that plasmids can pass through more readily. n Several techniques may be used to make bacterial cells competent: a common method uses calcium chloride. Charges are Important n Plasmid DNA is negatively charged (phosphate groups).
n Plasma membranes surrounding bacteria contain phosphate groups and are also negatively charged.
n To overcome the repelling force of the two negative electrical charges, a solution of calcium chloride will neutralize the negative charges.
Introduction of Calcium Introduce Ca2+ andlower temperature n Transferring samples from ice to warm water = "heat shock" n It is thought a slight pressure difference is created between the outside and the inside of the bacterial cell that makes the cell wall and membrane more permeable.
n The pressure moves plasmid into the bacterial cell.
Transformation & Colony Formation Formation n Bacteria are exposed to a defined n Only bacteria that take up copies of the plasmid survive (the plasmid makes them resistant).
n "Protecting genes" contribute to expression of proteins that break down the antibiotics. n The antibiotics act as a filter to select only the modified bacteria.
n Bacteria can be grown in large amounts, harvested, and lysed to isolate the plasmid/DNA of interest. Streaking Agar Plates Luria Bertani (LB) Medium n A nutrient-rich media commonly used to culture bacteria in the lab.
n Addition of agar to LB results in the formation of a gel that bacteria can grow on – bacteria cannot digest the agar but can gather nutrition from the LB within.
n Addition of an antibiotic to this gel allows for the selection of only those bacteria with the specific antibiotic resistance - usually conferred by a plasmid carrying the antibiotic resistance gene. n Media contains: Tryptone (enzymatically digested milk protein casein – source of amino acids) Yeast extract (source of nutrients) Glucose (energy source) Transferring Bacteria to Agar Plates Goal: to isolate
Commonly Used Antibiotics n Ampicillin (100 µg/mL) n Bleocin (5 µg/mL) n Carbenicillin (100 µg/mL)n Chloramphenicol (25 µg/mLn Coumermycin (25 µg/mL)n Gentamycin (10 µg/mL)n Kanamycin (50 µg/mL) n Spectinomycin (50 µg/mL)n Tetracycline (10 µg/mL) Successful Colony Formation n Using a single colony from a freshly streaked agar plate to inoculate a bacterial culture for DNA purification will minimize the chance of having a mixture of plasmids in your purified DNA. Innoculating a Liquid Bacterial Culture n Individual colonies can be isolated from an LB agar plate.
n A liquid LB culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers of bacteria necessary to isolate enough plasmid DNA for experimental use. High Copy vs. Low Copy Plasmids n Copy number refers to the number of copies of an individual plasmid within a single bacterial cell. n Large plasmids usually have a low copy number ( 1-2 copies per cell) and need to grow for longer periods of time ( 18-30 hr). n Smaller plasmids can be present in large numbers (50+ per cell; high copy number) but should only be grown for 12-16 hr.
n Certain features of a plasmid may render it low copy regardless of Recovering DNA From Bacteria n Many companies (i.e., Qiagen, Invitrogen, Promega, etc) sell kits for isolating plasmid DNA.
n Sensitivity: quantities as low as a few µg to as much as several mg; concentrations ranging from 150 ng/µl to several µg/µl. Protein Extraction Chromatography resins are used for affinity purification Proteins separated by molecular mass (electrophoresis) Bacterial – Plasmid Storage n Plasmid DNA can be stored at -20°C n Bacteria on an LB agar plate can be stored at 4°C for several n Glycerol stocks allow for long-term storage - addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive.
n A glycerol stock of bacteria can be stored stably at -80°C for Lab 1: Bacterial DNA & Protein Extraction n Each group will receive 2 mL of E.coli expressing C1-Venus, and 2 mL of E.coli expressing RFP.
n DNA plasmids will be extracted using purifcation columns.
n Plasmid purification will be confirmed by agarose gel n Protein extraction from lysing bacterial samples n PAGE (polyacrylamide gel electrophoresis) for protein separation to confirm protein isolation Your Samples are Precious! n You will use different methods to quantify your extracted DNA and protein during Lab 2.
n You will perform qPCR during Lab 3 with your extracted DNA sample.
n You will transfect mammalian cells with your DNA in Lab 5 – and confirm protein expression by microscopic observation.
Working in the IBBME Teaching Lab courtesy Richard Kwan n Located at MB 325 n Dr. Andrey Shukalyuk, Coordinator n Biosafety Level 2 facility = "includes agents that pose moderate hazards to personnel and the environment" Occupational Health & Safety Act (OHSA) n While working in the UG TL, you • know about workplace hazards and what to do about them; • participate in solving H&S problems;• refuse to work in the lab if you believe the environment is unsafe.
Your Responsibilities in the Lab n To work in the lab, you must: • follow the law and U of T/IBBME H&S policies and • wear and use protective equipment provided;• work and act in a responsible manner (safe lab practice);• report any hazards or injuries to your TA/Lab Coordinator The Responsibility of the TL: n Ensure you are aware about hazards and dangers and demonstrate how to work safely in the UG TL; n Ensure your TAs are know the IBBME Safety n Ensure you are wearing and properly using protective n Ensure you follow the law and workplace procedures; n Do everything reasonable (given any circumstance) to protect you from being hurt while working in the lab.
Know Your Environment n Familiarize yourself with the location of the nearest telephone, emergencytelephone numbers, first aid kit, spill kit,fire extinguisher, fire alarms, exits, etc.
n Know the location of eyewash stations, emergency showers and handwash stations.
n Report any accident to the TA/Lab Coordinator IMMEDIATELY.
n If there is a fire, use the fire extinguisher (small fire) or pull the alarm and calmly evacuate.
n Workplace Hazardous Materials Information System n A series of distinct symbols that quickly and intuitively alert a user to different types of hazards n U of T Environmental Health & Safety (EH&S) online training [EHS001] through the Blackboard Portal Chemical Use in the Lab n Always be careful when using laboratory chemicals n Multiple routes of entry: • inhalation – use fumes hoods• ingestion – do not eat/drink/chew gum in lab• injection – do not use broken or chipped glassware• via mucous membranes (i.e., eyes) – wear goggles n If you aren't sure, ASK! summary regarding procedures for safe handling, storage, and disposal of laboratory chemicals where to access (hard copy or electronically) Proper Waste Disposal Observe good housekeeping practices. Maintain clean and tidy work areas, particularly when you leave the Micou & Kilkenny, 2012 General Lab Safety n Place all personal effects into the drawers beneath the lab
benches (jackets, backpacks, cellphones, etc). n Wear proper personal protective equipment.
n Tie back long hair.
n Always wear long pants and closed-toe shoes.
Proper Glove Use n Remove jewellery n Inspect gloves first ensure no n Pull gloves on over cuffs of lab n Discard to appropriate waste when finished wearing (do not reuse) DO NOT wear PPE outside of the laboratory
(please do not remove lab equipment either) General Considerationsn Be careful not to touch your face or other personal items (such as your cellphone) with gloved hands. n Always wash your hands with soap and water at designated sinks before leaving the lab (even if you have been wearing gloves or you didn't do anything at all).
10 OF THE MOST COMMON HUMAN FACTORS THAT CAUSE ACCIDENTSNational Safety Council n NEGLIGENCE - failure to observe
n CURIOSITY - doing something
safety instructions or maintain
unexpected just to see what
n ANGER - causes one to become
n INADEQUATE INSTRUCTION -
irrational and disregard common
results in untrained or improperly
n HASTE - acting before thinking can
n POOR WORK HABITS - cluttered
lead people to make hazardous
floor or work area, loose clothing,
n INDIFFERENCE - lack of attention
n OVERCONFIDENCE - displayed by
to the task
behavior that is too cocky, risk
n DISTRACTIONS - interruptions by
taking, etc
others while performing routine or
n LACK OF PLANNING - not taking
The Laboratory Notebook n This is a permanent record of n Everything you do in the lab should be recorded in this book: strategy, planning, execution, results. n Another individual should be able to replicate yourexperiment based on whatyou have written.
Writing in the Notebook n Writing must be legible and in ink – NO PENCIL! n Do not erase anything, and do not remove pages. Errors should be crossed out.
n Everything should be handwritten. The only exceptions are data printouts from an instrument (i.e., spectrometer), a printed image, or a pre- printed standard form (i.e., a table).
n Never leave large gaps – this will prevent erroneous information from being added later.
n Use the first two pages as a Table of Contents.
What to Write In Advance n Begin each lab on a new page; pages should be consecutively n Each lab write-up should include the title of the experiment, date, partner's name (if applicable) and objective of the lab/protocol. n Methods flowcharts are a great way to ensure you've gone through the protocol and understand what you need to do.
n Sample calculations (i.e., required dilutions) should be recorded in the notebook before coming to the lab.
While Performing the Experiment: n Include any protocol deviations (intentional or accidental). n Record all observations, even things that seem trivial, in real- time - nothing should be added "after the fact".
n Include all data: raw and calculated. n Discussion – interpret your results .
n Draw conclusions about your experiment.
n Make suggestions for future experiments or protocol When the Lab Session is Finished n Initial each notebook page at the bottom. n Make sure your TA initials the data page. n Provide the duplicate copy to your TA before you leave the lab. Make sure the TA can read the copy! n Post-lab questions can be answered at the end of the data collection, or on a separate page with data analysis.
n Results and post-lab answers are to be handed in at the beginning of the next lab session. Late submissions will NOT
be accepted.
Basics of Pipetting n Liquid transfer depends upon liquid volume, liquid viscosity, sterility requirements and required accuracy. n Commonly-used equipment includes: • graduated cylinders • electric Pipette-aids + serological pipettes • pasteur pipettes • micropipettes (single channel, multichannel, repeater) n Piston-driven air displacement n Range 0.5 – 1000 μl n % error ranges from 0.5-5 depending upon micropipette and desired volume n Typically named after volume delivered (i.e., a "P200" is capable of delivering up to 200 μl) General Micropipette Guidelines n Select the correct pipette for desired liquid volume (10% n Select the correct tips and attach snugly. n When aspirating liquid, submerge tip only slightly beneath n When dispensing liquid, hold pipette tip against inner surface of receptacle to allow smooth delivery. n Never allow liquid into the pipette barrel. n Press plunger smoothly. n Change tip after each use (discard as appropriate). Micou & Kilkenny, 2012 Forward Pipetting n Technique used for non-viscous (aqueous) liquids. n Depress plunger to first stop and release to draw liquid into n Wipe tip against side of container to remove excess. n Press plunger to first stop to deliver liquid. n Pressing plunger to second stop will release tip. Reverse Pipetting n Technique for viscous or foamy liquids, or small liquid n Press plunger to second stop and draw liquid into tip. n Wipe tip against receptacle wall to remove excess. n To deliver liquid, release plunger to first stop and hold for 1-2 n Remaining liquid should be discarded. Relevant Terminology n Biotechnology refers to any technology used to manipulate
DNA (genetic engineering).
n Recombinant DNA refers to DNA from two different sources.

Source: http://engsci.andrewwong.tk/files/3S/BME346-L02.pdf