Molecular Biology Techniques. A Classroom Laboratory Manual

271 Pages·2019·18.21 MB·english

by  Susan Carson

Molecular Biology Techniques
Molecular Biology
Techniques
A Classroom Laboratory Manual
Fourth Edition
Susan Carson
Department of Plant and Microbial Biology, North Carolina State University,
Raleigh, NC, United States
Heather B. Miller
Department of Chemistry, High Point University, High Point, NC, United States
D. Scott Witherow
Department of Chemistry, Biochemistry, and Physics, The University of Tampa,
Tampa, FL, United States
Melissa C. Srougi
Department of Chemistry, High Point University, High Point, NC, United States
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About the Authors
Dr. Susan Carson is a Professor of Plant and Microbial Biology and leads academic enrichment programs and faculty
development in the area of enhancing students’ critical and creative thinking skills at North Carolina State University.
She graduated from Rutgers University (New Brunswick, NJ) with a BS in Biotechnology, and from the University of
North Carolina (Chapel Hill, NC) with a PhD in Microbiology. Her area of scientific expertise is in molecular mechan-
ismsofbacterialpathogenesis.Priortohercurrentrole,Dr.Carsonspentover10yearsleadingcurriculumdevelopment
for the NC State Biotechnology Program as its academic coordinator. Her scholarly work over the past 18 years has
focused on college-level biology education and enhancing students’ higher order thinking. She has received multiple
awards for teaching excellence and innovation and is a member of the Howard Hughes Science Education Alliance,
promoting and implementing inquiry-guided learning and authentic research in the undergraduate classroom laboratory.
She coauthored two molecular biology lab manuals and has published numerous peer-reviewed papers in the area of
course and curriculum development. She has mentored over 100 undergraduate students in research projects and is the
Principle Investigator and Director of the National Science Foundation (NSF)-funded Integrative Molecular Plant
Systems Research Experience for Undergraduates (REU) Program. She serves on the Leadership Council of the NSF
BIOREUandhasservedontheBoardofDirectorsoftheWakeCountyBeekeepingAssociation.
Dr.HeatherB.MillerisanAssistantProfessorofBiochemistryatHighPointUniversity(HighPoint,NC).Shegradu-
ated from Clarion University of Pennsylvania (Clarion, PA) with a BS in Molecular Biology/Biotechnology, and from
Duke University (Durham, NC) with a PhD in Molecular Genetics and Microbiology. She completed a teaching post-
doctoral position in the Biotechnology Program at North Carolina State University. Her area of scientific expertise is
RNA biology. Her research has focused on HIV-1 and MRSA gene expression. She has developed and taught multiple
biochemistry and biotechnology courses and has published and presented a number of peer-reviewed papers in the
scholarshipofteachingandlearning.
Dr. D.Scott Witherow is anAssociateProfessor ofBiochemistryat TheUniversityofTampa(Tampa,FL).He gradu-
ated from Rollins College (Winter Park, FL) with an AB in Chemistry and from the University of Miami (Miami, FL)
withaPhDinMolecularandCellularPharmacology.HisresearchhasfocusedprimarilyontheregulationofG-protein-
mediated signal transduction processes and biochemical education and pedagogy. He has been recognized by the
AmericanSocietyfor Biochemistry andMolecular Biologyasaneducationfellow forhis efforts inthe areas ofinstruc-
tionandassessment.
Dr.MelissaC.SrougiisanAssistantProfessorofBiochemistryatHighPointUniversity(HighPoint,NC).Shegradu-
ated from the University of Toledo (Toledo, OH) with a BS in Biology and from Case Western Reserve University
(Cleveland, OH) with a PhD in Pharmacology. Her scientific areas of expertise are in experimental cancer chemothera-
peutic agents and mechanisms of chemotherapeutic resistance. She actively trains undergraduate research students in
her laboratory. She co-founded and directs the High Point University Mobile Community Lab to facilitate the accessi-
bility of science to the lay community. In addition, she has developed and taught a variety of inquiry-based, college-
level science courses and has published and presented a number of peer-reviewed papers in the scholarship of teaching
andlearning.
xi
Preface
Recombinant DNA technology touches many aspects of our lives. From drought-tolerant crops to biofuels to pharma-
ceuticals, genetically modified organisms surround us. Throughout its history, biotechnology has relied heavily on
DNA cloning to produce protein products more efficiently or to make modified genes or combinations of genes.
Escherichia coli or other hosts serve as cellular “factories” to churn out large amounts of protein. Human insulin, for
example,isproducedrecombinantlyusingmanyofthesametechniquesdescribedinthisbook.
In the eight years since the third edition of Molecular Biology Techniques was published, molecular biology
researchanditstoolshaveundergonemajortransformationsandcontinuetoevolve.
Modulatinggeneexpressionhasbecomeamainstayinmanylaboratories.RNAiandCRISPR,especially,haverevo-
lutionized the way scientists study gene function, as well as design selective therapies for various disease states. As
thesetechnologiesarebecomingacornerstoneinallfacetsofmodernmedicineandbiotechnology,studentswillbenefit
from a working knowledge of their theory and methods. Therefore, this expanded classroom laboratory manual now
includes laboratory sessions on mammalian cell culture, RNAi, and CRISPR for use as a stand-alone, semester-long
projectforanadvancedcourseorinconjunctionwiththeprevioussessionsasatwo-semestersequence.
What will we learn in the next eight years? Students taking this course right now will be at the leading edge of
excitingnewdiscoveries.Wecan’twaittofindout!
xiii
Acknowledgments
WewouldliketothankthemanypeoplewhocontributedtothiseditionofMolecularBiologyTechniques.Asthisproj-
ect has grown over the years, so have the number of individuals who made this work possible. First, we would like to
thank our editorial team at Elsevier for bringing this work to life. Dr. Sam Wolff at the University of North Carolina at
Chapel Hill was instrumental in his suggestions for CRISPR experimental design. High Point University biology, bio-
chemistry, and chemistry students were helpful in piloting the CRISPR laboratory sessions and providing feedback on
the previous edition. Students from The University of Tampa were helpful in providing feedback on the previous edi-
tionandpilotingsomeoftheadvancedexperiments.WewouldalsoliketothankHighPointUniversityCollegeofArts
and Sciences and the Department of Chemistry, as well as The University of Tampa College of Natural and Health
SciencesandtheDepartmentofChemistry,Biochemistry,andPhysicsforsupportingclassesthatusethisbook.Finally,
theauthorsacknowledgetheirspousesandchildrenfortheirunwaveringsupportduringthepreparationofthisbook.
xv
Note to Instructors
This lab manual was originally developed in the context of the curriculum offered by the North Carolina State
University Biotechnology Program. Over the past decade, there have been numerous advances in molecular biotechnol-
ogy that have changed how scientists test hypotheses. Despite these advances, many common techniques still remain
the same. In this 4th edition, we have added a new section of material dedicated to some of these newer techniques,
most notably CRISPR, which was named Science Magazine’s Breakthrough of the Year in 2015. The laboratory ses-
sionsdescribedinthismanualaredesignedtopreparestudentsformorespecializedupper-levelcoursesinbiochemistry
andmolecularbiologyandforindependentresearchprojectsatboththeundergraduateandgraduatelevel.
A typical course using this manual meets at least 4hours for laboratory per week coupled with several (B30min-
utes)interimlaboratorysessionsthroughoutthesemester.WerecommendthisscheduleforPartsI(cid:1)IVbecause,forsev-
eral of the lab exercises, students must inoculate cultures or perform other short activities prior to their lab day. For
classes performing cell culture experiments (Part V: Modulation of Gene Expression), it is also useful to meet more
than once a week. Maintaining cells requires careful monitoring and maintenance, dictating the need for interim labora-
tory sessions. We have alerted instructors in the text that certain steps must be performed at particular times either by
theclassasaninterimlaboratorysessionorbytheinstructororteachingassistant,“behindthescenes.”
The majority of the laboratory exercises do not require the full 4hours to complete for students who come to lab
prepared. Additionally, there are a few labs for which incubation times are simply too long to reasonably include in the
exercises. Inthese cases, the steps are included in the protocols with a note that the instructor will perform that particu-
lar part of the experiment for the class (for example, the induction of the fusion protein using IPTG, necessary for sev-
eral laboratories, takes 2(cid:1)4hours). For this reason, we recommend offering the laboratory as an afternoon course so
thattheinstructorcanbegintheincubationsinthemorning,ratherthanthemiddleofthenight.
We are aware that earlier editions of this manual are being used at a wide range of institutions and levels, ranging
from prep schools to graduate programs. While our aim is to design the experiments towards introductory molecular
biology/biochemistry students, included in this edition are a number of Advanced Alternatives. These alternatives are
written primarily for upper-level undergraduates or graduate students who have previous laboratory experience with
many of the basic techniques described in the regular lab sessions. These alternatives include whole project ideas, such
as cloning your own gene of interest, to more specific alternatives, such as altering the design of a western blot to
enableittobeperformedsemi-quantitatively.ThesealternativescanbefoundatthebeginningofPartsI(cid:1)III.
Parts I(cid:1)III comprise of a 12-week course suitable for an introductory molecular biology or molecular biochemistry
lab. These experiments include PCR cloning enhanced green fluorescent protein (egfp) cDNA into a bacterial expres-
sion vector (Part I: DNA Manipulation); screening for positive clones (Part II: Screening Transformants); and expres-
sion, purification, and quantification of the recombinant protein (Part III: Expression, Detection and Purification of
Recombinant Proteins from Bacteria). The analysis and quantification of mRNA is described in Part IV, which can be
done in two to three additional lab sessions. For a typical 15-week course, one can fit in all the experiments described
inPartsI(cid:1)IV,althoughmanyinstructorschoosetofocusjustonthefirst12labs,leavingtimeforotherclassroomexer-
cises, presentations, etc. Part V, Modulation of Gene Expression, is new to this edition and focuses on controlling gene
expression in mammalian cells. These lab sessions are designed to provide students with the background theory and
workingknowledgenotonlyonhowtoculturemammaliancells,butalsoonhowtoaltergeneexpressioninthismodel
system using transient transfection, RNAi, and CRISPR technologies. These experiments are best performed within a
separate course. The most important thing to note is all the lab sessions outlined in this manual are not designed to be
implemented in a single semester. However, over the course of two semesters, there are a number of ways to perform
alltheexperiments,dependingonyourspecificcurricularneeds.
Throughoutthetext,wehaveaddedbothtime-savingandmoney-savingtips.Thesesuggestionsareincludedtohelp
instructorsusetheirresourceswisely.
xvii
xviii NotetoInstructors
In preparing students for potential laboratory work after graduation, it may be helpful to introduce a virtual (elec-
tronic) lab notebook rather than a typical bound notebook. We have found these to have many advantages. For one, the
instructor can have access to student notebook entries 24/7 from his or her computer. For the student, the virtual lab
notebook enables one to simply upload gel or blot images, Excel spreadsheets, and more, rather than printing, cutting,
andtaping into aboundnotebook.Theycan alsoquickly create tables,checklists,andprotocols.These notebookselim-
inate the problem of illegible handwriting and lost notebooks. In addition, some have incorporated cloning, oligo,
sgRNA design, and sequence analysis tools. Entries can still be printed, as well. Benchling (http://www.benchling.com)
is one version that is freely available. LabArchives is also popular (http://www.labarchives.com), but there is an associ-
atedfeeforuse.
For instructors implementing the experiments described in Part V, Modulation of Gene Expression, these labs
require a Biosafety Level 2 (BSL-2) laboratory space for working with HEK293 cells. Before engaging in these experi-
ments, please ensure that you have the requisite approval through your institution’s biosafety organization. BSL-2
guidelines can be found through the Centers for Disease Control and Prevention (http://www.cdc.gov/biosafety/publica-
tions/bmbl5/index.htm);notablySectionIV—LaboratoryBiosafetyLevelCriteria.
We recommend using commercially prepared competent Escherichia coli for the experiments in this course, as they
tend to perform consistently from batch to batch. If your budget does not permit this, it is possible to prepare “home-
made” competent cells and store them in a 280(cid:3)C freezer. We have included the protocol for preparation of competent
cellsintheAppendix.
Allantibodiesdescribedinthismanualareavailablecommercially.ThepBITandpEGFP-N1plasmidsareavailable
at no cost to institutions of higher education for educational purposes from Dr. Scott Witherow at The University of
Tampa. Contact Dr. Witherow at [email protected] and include in the subject heading “pBIT request.” pET-41a,
pLentiCRISPR-E, and pLentiCRISPR-EGFP are available commercially, and we are not licensed to distribute them.
HEK293 mammalian cells, as well as HEK293 cells stably expressing GFP, can be purchased commercially for use in
PartV,ModulationofGeneExpression.
Nomenclature
In the literature, the nomenclature for the abbreviations of the enhanced green fluorescent protein gene and its gene
product has been inconsistent, at best, and downright confusing at worst. In this publication, we will use “egfp” to refer
to the gene (either DNA or mRNA) and “EGFP” to refer to the gene product. Likewise, we will use “gst” for the
glutathione-S-transferase gene and “GST” for its gene product. Bacterial genes discussed in this book will use standard
bacterial nomenclature with the gene name lowercase italicized, and the gene product with a capitalized first letter and
notitalicized.Forexample,thegeneforthelacrepressoris“lacI”anditsgeneproductis“LacI.”
xix
Introduction
CONCEPTUAL OUTLINE FOR EXPERIMENTS
Parts I, II, and III of this manual describe the process of making a fusion protein by joining genes from two organisms:
one from Escherichia coli (gst) and an enhanced gene derived from the green fluorescent jellyfish Aequorea victoria
(egfp). Expression of the fused gene will produce a single protein in bacteria, which can be readily purified due to the
GSTportionofthefusionprotein.Thepurifiedproteincanbevisualizedbyfluorescenceandquantified.
Part IVmovesbeyond theprotein level tostudy mRNA levels usingreversetranscription PCR (RT-PCR). Levelsof
mRNAinthepresenceandabsenceofinducingmoleculescanbequantifiedandcompared.
In Part V, we leave the bacterial system and move into mammalian cell culture. In this part of the manual, EGFP is
constitutively expressed in a mammalian cell line and gene expression modulated using two different methods: RNAi
andCRISPR.
In summary, the various parts of this manual are designed to explain the theory, outline the experimental design,
and guide in the analysis of the data for common biochemical and molecular biology techniques used in many research
laboratories.
EXPERIMENTAL PROCEDURES
Part I: Manipulation of DNA
(SeeFig.1foradiagrammaticrepresentation.)
(cid:1) IsolateplasmidDNAusingculturesofbacteriacontainingtheE.coliexpressionvectorpET-41a.
(cid:1) UserestrictionenzymestocutthepET-41avector.
(cid:1) UsePCRtoamplifytheinsert(A.victoriaegfpDNA)frompEGFP-N1,andrestrictiondigesttoformstickyends.
(cid:1) UseDNAligaseto“paste”thevectorandinsertDNAtogether.
(cid:1) IntroducetheligatedDNAintoE.coli.
Part II: Screening Transformants
(cid:1) Confirmpositiveclonescontainingthegst::egfpDNAbypolymerasechainreaction.
(cid:1) Isolate DNA from transformants and digest with restriction enzymes to further validate the presence of the gst::egfp
fusionDNA.
(cid:1) ConfirmEGFP-positiveclonesbyfluorescence.
(cid:1) VerifythatsinglenucleotideerrorsdidnotoccurintheegfpgeneduringPCRcloningbyDNAsequencing.
Part III: Expression, Detection, and Purification of Recombinant Proteins From Bacteria
(cid:1) Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS(cid:1)PAGE) and western blot analysis to confirm
theexpressionofthefusionprotein.
(cid:1) Induce a large-scale culture of the transformed bacteria with isopropyl-β-D-thiogalactopyranoside (IPTG) to make
largeamountsofthefusionprotein.
(cid:1) Purifythefusionproteinonasubstrateaffinitycolumn.
(cid:1) Performproteinquantificationofelutedfractions.
(cid:1) UseSDS(cid:1)PAGEofpurificationfractionstocheckforpurityanddegradation.
xxi