Massachusetts Institute of Technology
Department of Biology
31 Ames Street, Cambridge, MA
Molecular Biology Techniques
IAP 2007
Course 7.391
Jan 8-12, 16-19, & 22
12:30 PM - 4:30 PM
Building 68-230
3 U credits
Prepared & Taught by
MANDANA SASSANFAR, Ph.D.
&
PETER WEIGELE, Ph.D.
Funded by the Howard Hughes Medical Institute
Synopsis of the methods
taught in this course:
Laboratory safety
Pipetting
Sterile techniques
Bacteriophage plating and propagation
Measurement of viral titer by serial dilution
Column chromatography: anion exchange and affinity based
CsCl density gradient ultracentrifugation
Plasmid and genomic DNA purification
Agaraose gel electrophoresis of DNA
SDS-polyacrylamide gel electrophoresis of proteins
Oligonucleotide design and polymerase chain reaction (PCR)
Gene cloning using PCR, restriction digest, and ligation
Analysis of DNA sequences: sequencing, restriction digests
and database searching
Transmission electron microscopy (TEM)
TABLE OF CONTENTS
Synopsis of methods i
Summary ii
About the course instuctors
Laboratory safety
Techniques
Oveview of laboratory and experimental schedule iii
Introduction and backround 1
Day 1: Concentration and purification of freshwater phage communities
Day 2: Isolation of phages on bacterial hosts
Day 3: Determining phage titers
Day 4:Large scale purification of phages
Day 5:
Day 6:
Day 7:
Day 8:
Day 9:
Day 10:
Appendix 1:
Appendix 2:
Appendix 3: Plasmid maps
Appendix 4: Reading a Scientific Paper
Tables
Acknowledgements
Finding out More!
Summary
In this short, but intense, two week laboratory course, we will learn and use a collection of laboratory techniques to accomplish a single research goal: the isolation, characterization and molecular dissection of a virus (which you will isolate). The techniqes learned in this course will provide a repertoire of methods you will be able to use in any lab practicing traditional microbiology.
You will begin by concentrating whole virus communities out of water samples using anion exchange chromotography. Different bacterial strains will be incubated together with these virus concentrates on the suface bacterial medium solidified with agar in a Petri dish. If any of the bacteria are susceptible to the viruses in the concentrate, a clear spot (plaque) caused by a zone of killing by the virus will be seen. The viruses in this plaque will be subsequently propagated in large liquid cultures and purified using a series of biochemical techniques. The proteins and DNA of these viruses will be extracted and examined by gel electrophoresis. The morphology of your virus will be observed directly using a transmission electron microscope. At this stage, your virus would be ready for genomic sequencing. However, due to time limitations, we will subsequently use gene sequences from viruses that have already been sequenced. You will learn how to go from raw sequence data to annotated genome using a series of online tools that include identificaiton of open reading frames (protein coding sequences) and identification of known homologes in the sequence database.
A set of genes from our collection of aforementioned viruses whose genomes have been sequenced will be the target of PCR cloning. This same set of protocols could be used to clone a gene from the virus you isolated if you knew already the gene's nucleotide sequence. The genes will be first amplified from the genomic DNA templates using sequence specific oligonucleotide primers and the polymerase chain reaction. The resulting PCR product will have its ends cut with restriction enzymes and be ligated to a plasmid vector similarly cut to yield cohesive compatible (“sticky”) ends. This plasmid vector contains both promoter sequences to control the expression of inserted gene, as well as a antibiotic resistance gene that serves as a selectable marker ensuring that host bacterial cells maintain the plasmid through successive generations. The ligation mixture containing successful as well as failed ligation events will be used to transform competent bacterial cells to antibiotic resistance. Resulting transformants will be screened for plasmids that successfully contain the inserted gene. Plasmids that appear to have the insert will be confirmed by DNA sequencing.
About your instructors
Dr. Mandana Sassanfar- Mandana has been an instructor in the biology department at MIT since July 2002. In the summer, she directs a summer research program for undergraduates from other college, and organizes and teaches a workshop for AP biology and honors biology teachers from the Boston area. In the spring she teaches an undergraduate seminar and organizes an MIT field trip for high cshool students. In the fall she recruits students for the biology PhD program.
Mandana earned her B.S and M.S in Biochemistry from the University Pierre et Marie Curie in Paris (she has indeed a French accent). She earned her Ph.D. degree in biochemistry from Cornell University where she worked on DNA damage in E. coli. She really enjoyed leaving in Ithaca. She then came to Boston where she first completed postdoctoral work at the Harvard School of Public Health on DNA alkylation damage and DNA repair in yeast and human, and then in the Department of Molecular Biology at MGH where she worked on RNA aptamer selection. She then did a 4-year stint in a start up biotech company in Cambridge selecting for new bacterial drug targets associated with protein syntesis, before returning to academia, first at Harvard and then at MIT.
Dr. Peter Weigele- Peter is a Research Scientist in the laboratory of Prof. Jonathan King. The King Laboratory's main focus is protein folding and experiments using the P22 tailspike protein (a phage protein) and the crystallins (components of the eye lens involved in transparency and cataract) provide researchers insights into how the linear amino acid sequence of a protein governs its three-dimensional shape. The King lab also has projects aimed at determining the structure of a phage in the act of injecting its DNA as well understanding the physiology of cyanobacteria (bacteria which can do oxygenic photosynthesis) infected by bacteriophage.
Peter started his scientific career doing undergraduate research in a fruit fly lab at the State University of New York at Albany. After graduation and a two year stint as a laboratory technician, he went to graduate school at the University of Utah. In 2001, he completed his doctoral dissertation on the morphogenesis of viral capsids under the mentorship of Prof. Sherwood Casjens. Peter joined the Department of Biology at MIT in January, 2002, as a post-doctoral associate. Currently, he is Research Scientist and Microscope Manager for the Biological TEM Users Group. In addition to his research, Peter volunteers at the Edgerton Center through the activities of the Biological Energy Interest Group (BEInG) which he helped form in 2004. The BEInG has developed an algal fuel cell as a low-cost platform for research and education in bioengineering for sustainability at the undergraduate level.
WELCOME to 2007 IAP
The Labs will be held daily from 12:30 PM to 4:30 PM in room 68-246
Please arrive on time (as early as 12:15 PM if possible). We will begin at 12:30 PM each day with an introduction and discussion of the agenda for the afternoon.
You will be working in teams of 3.
Each session will include:
· Introduction to the afternoon's experiments
· Setting up for the experiments
· Reviewing the previous day's experiments
· Analyzing the data obtained from the previous day's experiments
· Preparing for next day's experiments; e.g., inoculating overnight cultures and properly putting away/storing reagents and strains overnight.
LABORATORY SAFETY RULES
· ABSOLUTELY NO EATING OR DRINKING IN THE LAB!
· Wear a lab coat at all time in the lab. Do not wear open-toe shoes
· Wear gloves when handling chemicals
· Dispose of all bacterial culture and contaminated liquids in 10% bleach.
· Rinse all flasks and beakers with water before putting in the wash
· Seal used agar plates with parafilm and dispose in biohazard autoclavable orange bags
· Dispose of all tips, and sharps in appropriate red containers
· Keep benches clean and free of personal belongings at all time
· Wipe bench with 70% ethanol before and after each experiments.
· Wash hands with soap or alcohol-based hand sanitizers
· Dispose of all pipettes and reagents in appropriate containers.
· Keep written records of any observation and any deviation from protocols.
· Record type of equipment used (name, brand, model) and name of supplier.
· Avoid contamination of the plates and bacterial cultures by working under sterile conditions
· Store all plates, phage stocks and phage dilution at 4ºC
TECHNIQUES
Overview
This 2-week class will introduce you to some basic laboratory skills such as keeping a laboratory notebook, preparation of reagents and media, lab safety, how to correctly dispose of chemicals and lab etiquette (cleaning up after yourself J) as well as basic laboratory techniques for handling bacteria, DNA and proteins.
You will be working with four species of bacteria (E.coli, Vibrio, Corrynebacterium, and Salmonella) and an unknown mixture of aquatic phages that you will concentrate from water samples collected from the Charles River at the MIT boat house as well as the MWRA Deer Island sewage treatment facility. We will use microbiology, sterile techniques, ion-exchange chromatography, SDS-PAGE, DNA restriction analysis, DNA sequencing, electron microscopy, and bioinformatics tools to grow, purify, and characterize the phages found in these samples. We will also use PCR to amplify and clone a specific phage gene into a protein expression vector.
Microbiology
You will learn how to concentrate and isolate novel phages from water samples, how to maintain and store bacterial strains, how to streak for single colonies on agar plates, inoculate liquid cultures with single bacterial colonies -all under sterile conditions-, how to plate phage samples on bacterial host, how to perform a plaque assay, how to prepare sterile solutions, how to determine the pfu ( plaque forming unit) of a phage stock, and how to store phages for long periods of times. You will also pour your own agar plates and prepare buffers from stock solutions.
Protein and DNA analysis
After preparing large quantities of the phage you will estimate how many types of proteins are present in the phage capsids by preparing an SDS polyacrylamide gel and using it to separate the proteins of the capsid by size. You will stain and destain the gel and analyze the results.
You will also isolate phage DNA from a single phage strain, digest the DNA with various restriction enzymes and view the results on an agarose gel. You will use the results to determine the diversity of the phage population and to estimate the size of the phage genome. You will learn how to visualize the molecules of proteins or DNA, and how to record and analyze you data.
PCR and cloning
You will be given the sequence of a phage gene and learn how to design primers to clone the gene into a given vector. You will first amplify the desired gene sequence by PCR using those primers, purify the PCR fragment, digest it with restriction enzyme and clone it into a vector by ligation and bacterial transformation. You will then again use PCR, this time to verify that you have the right size insert in the vector.
Bioinformatics
You will also learn some basic bioinfomatics and sequence data analysis. These tool will help you in the correct design of oligonucleotide primers for PCR, verification of DNA sequences, and database homology searching.
LABORATORY SCHEDULE
NOTE: It takes at least 4 to 5 days to start from a water sample to isolate, purify and amplify enough phages for further characterization. Therefore most of the first week will be spent doing microbiology, and isolating enough phages for the second week’s experiments. We will also do a protein purification and a PCR cloning in the first week. The second week will mostly focus on the characterization of the phages that you isolated and amplified the first week.
Note: Several experiments will take place simultaneously. The color code allows you to follow one experiment across several days.
The green color code is for bacterial culture. It is somewhat related to the purple-coded experiments in that you need to use fresh cultures of bacteria whenever needed and therefore must make sure to remember to set up overnights cultures every day before you leave.
Day 1:
· Safety guidelines
· Lab tour
· Overview of the course
· How to keep a notebook
· Weigh DE-52 cellulose and mix with water sample, set up column, pack cellulose into column, wash, let stand in cold overnight
· Aseptic techniques
· Inoculate overnight liquid cultures form bacterial plates (4 strains in total). Re-streak bacteria on solid medium (agar plate) for single colonies
Day 2:
· Elute phages of Column and collect fractions
· Determine protein concentration of fractions by the Bradford assay
· Pool fractions containing phages together
· Plate phages on each bacterial strain using low salt LB and top agar
· PCR part I. Oligonucleotide primer design for cloning
· Inoculate overnight liquid cultures from single bacterial colonies
Day 3:
· Look for the presence of plaques on Bacterial lawn. Record number and shape
· Purify phages from well-isolated single plaques (using a sterile Pasteur pipette). Treat with chloroform, let sit in cold room for couple hours
· Prepare 10-fold serial dilutions tubes for phage titering
· Plate phage dilutions 103, 104,105 and 106 using top agar and O/N bacterial cultures. Incubate overnight at 30 ºC
· PCR part 2: Set up the PCR reactions
· Set up overnight cultures from single bacterial colonies
Day 4:
· Record number of phage plaques for the various dilutions and strains
· Pick 2 to 4 well isolated plaques from the high dilution plates and place in 1 ml of phage buffer; add a few drops of chloroform, mix and let sit in cold for a few hours to elute phage.
· PCR: Analysis of PCR products by agarose gel electrophoresis
· Set up overnight cultures of bacterial hosts (for large scale preparation of virus)
Day 5:
· at 9 AM: set up 1/50 or 1/100 dilutions in LB of overnight bacterial culture and incubate at 37ºC with vigorous shaking. Monitor growth at OD600. using a spectrophotometer and disposable cuvettes.
· When OD600 of liquid cultures reaches between 0.25 and 0.3, infect cultures with phages eluted from the plaques.
· PCR part 3: Digest PCR products, ligate into vector, transform competent E. coli cells, plate and incubate O/N
· When the bacterial culture clears (most of the bacteria have been infected and lysed), add chloroform, mix, and centrifuge. Collect supernatant. Add solid NaCl and PEG. Incubate in cold room for at least 30 minutes (or until ready to set up the CsCl gradient ultracentrifugation).